Schizochytrium fatty acid synthase (FAS) and products and methods related thereto

ABSTRACT

Disclosed are a fatty acid synthase (FAS) from  Schizochytrium , biologically active fragments and homologues thereof, a nucleic acid sequence encoding such FAS, fragments and homologues thereof, the gene encoding  Schizochytrium  FAS, host cells and organisms that recombinantly express the FAS, host cells and organisms in which the expression and/or activity of the endogenous FAS has been attenuated, and various methods for making and using any of these proteins, nucleic acid molecules, genes, host cells or organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 11/058,046, filed on Feb. 14, 2005, now US Publication No.2005/0191679, which claims priority under 35 U.S.C. §119(e) from U.S.Provisional Application Ser. No. 60/544,692, filed Feb. 13, 2004. Theentire disclosures of each of the foregoing applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention generally relates to a fatty acid synthase (FAS) fromSchizochytrium and to products and methods related thereto.

BACKGROUND OF THE INVENTION

The de novo synthesis of short and medium chain saturated fatty acidsfrom acetyl-CoA and malonyl-CoA is a complex process catalyzed byseveral enzyme activities. In most bacteria and in plants theseactivities are associated with discrete monofunctional polypeptides. Infungi and animals however, these activities are integrated into one ortwo multifunctional polypeptide chains. Fatty acids, in particular inthe form of oils and fats, which are glycerol esters, play a major rolein human nutrition because of their high energy content. Additionally,specific types of fatty acids (e.g., polyunsaturated fatty acids such asDHA) have a wide range of physiological effects. There is great interestin being able to manipulate specific types of fatty acids made inorganisms that produce these fatty acids (e.g., in the form of oilsand/or phospholipids).

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated proteincomprising an amino acid sequence selected from: (a) an amino acidsequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequenceconsisting of positions 1-500 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 3900-4136 of SEQ ID NO:2, and a biologicallyactive fragment thereof; and (b) an amino acid sequence that is at leastabout 45% identical to any of the amino acid sequences of (a) and havingthe biological activity of the amino acid sequence of (a). In otheraspects of this embodiment, the isolated protein comprises an amino acidsequence that is at least about 60% identical, at least about 80%identical, or at least about 95% identical to any of the amino acidsequences of (a). In a preferred aspect of this embodiment, the proteincomprises an amino acid sequence selected from: an amino acid sequenceselected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting ofpositions 1-500 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3900-4136 of SEQ ID NO:2, or biologically active fragments ofany of these sequences. In an even more preferred embodiment, theprotein comprises an amino acid sequence selected from: SEQ ID NO:2, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:1, SEQ ID NO:12, and SEQ ID NO:13.

In one aspect of this embodiment, the protein comprises any two or moreamino acid sequences selected from the group consisting of: SEQ ID NO:2,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In another aspect,the protein comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ IDNO:13. In yet another aspect, the isolated protein is from aThraustochytriales microorganism and in a preferred embodiment, from aSchizochytrium microorganism.

Yet another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence encoding any ofthe above-identified proteins, or a nucleic acid sequence that is fullycomplementary thereto. Another embodiment of the present inventionrelates to a recombinant nucleic acid molecule comprising such anisolated nucleic acid molecule, operatively linked to a transcriptioncontrol sequence. In one aspect, the transcription control sequence is atissue-specific transcription control sequence. In another aspect, therecombinant nucleic acid molecule further comprises a targetingsequence. Yet another embodiment of the present invention relates to arecombinant cell that has been transformed with such a recombinantnucleic acid molecule.

Another embodiment of the present invention relates to a geneticallymodified microorganism for producing short chain fatty acids by abiosynthetic process, the microorganism being transformed with arecombinant nucleic acid molecule as described above.

Another embodiment of the present invention relates to a geneticallymodified plant for producing short chain fatty acids by a biosyntheticprocess, the plant being transformed with a recombinant nucleic acidmolecule as described above.

Yet another embodiment of the present invention relates to a geneticallymodified microorganism for producing short chain fatty acids by abiosynthetic process. The microorganism comprises a nucleic acidmolecule encoding a fatty acid synthase, wherein the nucleic acidmolecule has been modified to increase the expression or biologicalactivity of the fatty acid synthase. The fatty acid synthase comprisesan amino acid sequence selected from: (a) an amino acid sequenceselected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting ofpositions 1-500 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3900-4136 of SEQ ID NO:2, and a biologically active fragmentthereof; or (b) an amino acid sequence that is at least about 45%identical to any of the amino acid sequences of (a) and having thebiological activity of the amino acid sequence of (a). In oneembodiment, the nucleic acid molecule encoding a fatty acid synthase isan endogenous gene in the microorganism. In another embodiment, themicroorganism has been transformed with a nucleic acid molecule encodingthe fatty acid synthase. In yet another embodiment, the microorganismcomprises an endogenous gene encoding the fatty acid synthase and hasbeen transformed with a recombinant nucleic acid molecule encoding afatty acid synthase. In this embodiment, one or both of the gene and therecombinant nucleic acid molecule has been modified to increase theexpression or biological activity of the fatty acid synthase. Suchgenetically modified microorganisms can include Thraustochytrialesmicroorganism, and in one aspect, a Schizochytrium microorganism.

Another embodiment of the invention relates to a biomass comprising thegenetically modified microorganism described above, to a food productcomprising such a biomass, or to a pharmaceutical product comprisingsuch a biomass.

Another embodiment of the present invention relates to a method toproduce short chain fatty acids by a biosynthetic process, comprisingculturing in a fermentation medium a genetically modified microorganismas described above. Another embodiment of the present invention relatesto a method to produce short chain fatty acids by a biosyntheticprocess, comprising growing a genetically modified plant that has beentransformed with a recombinant nucleic acid molecule as described above.

Yet another embodiment of the present invention relates to anoligonucleotide, comprising at least 12 contiguous nucleotides of SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:4, or a nucleic acid sequence fullycomplementary thereto.

Another embodiment of the present invention relates to a geneticallymodified microorganism with reduced production of short chain fattyacids, wherein the microorganism has been genetically modified toselectively attenuate a fatty acid synthase gene or portion thereofencoding a functional domain. The fatty acid synthase gene comprises anucleic acid sequence selected from: (a) a nucleic acid sequenceencoding SEQ ID NO:2; and (b) a nucleic acid sequence encoding an aminoacid sequence that is at least about 45% identical to SEQ ID NO:2,wherein the protein having the amino acid sequence has a biologicalactivity selected from the group consisting of acetyl-transferase (AT)activity; enoyl ACP reductase (ER) activity; dehydrase (DH) activity;malonyl/palmitoyl acyltransferase (M/PAT) activity; a first acyl carrierprotein (ACP) activity; a second acyl carrier protein (ACP) activity;keto-acyl ACP reductase (KR) activity; keto-acyl ACP synthase (KS)activity; and phosphopantetheinyl transferase (PPT) activity. In oneaspect, the fatty acid synthase gene comprises a nucleic acid sequencerepresented by SEQ ID NO:1. In another aspect, the microorganism hasincreased production of at least one polyunsaturated fatty acid (PUFA).The microorganism can include, but is not limited to, aThraustochytriales microorganism, and particularly, a Schizochytrium. Inone aspect, the fatty acid synthase gene has been modified in aregulatory region to reduce expression of the gene. In another aspect,the fatty acid synthase gene has been modified in the coding region toreduce the biological activity of one or more functional domains of thefatty acid synthase. In yet another aspect, the fatty acid synthase genehas been mutated by targeted homologous recombination with a nucleicacid sequence that hybridizes to the fatty acid synthase gene andincludes a heterologous nucleic acid sequence that modifies the codingregion of the fatty acid synthase gene to reduce the expression oractivity of the fatty acid synthase encoded thereby.

Another embodiment of the invention relates to a biomass comprising thegenetically modified microorganisms described directly above, whereinthe microorganisms have reduced production of short chain fatty acids ascompared to a wild-type microorganism of the same species. Also includedin the invention are food products and pharmaceutical productscomprising such a biomass.

Yet another embodiment of the present invention relates to a method forincreasing the production of polyunsaturated fatty acids (PUFAs) in abiosynthetic process. The method includes the step of culturing underconditions effective to produce lipids comprising the PUFAs, geneticallymodified microorganisms as set forth directly above. Products comprisingthe lipids produced by such a method, including food products andpharmaceutical products, are also encompassed by the present invention.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIG. 1 is a schematic representation of the putative enzymatic domainspresent in the Schizochytrium FAS protein.

FIG. 2A is a schematic representation of the construction of a plasmidused for targeted inactivation of the Schizochytrium FAS gene.

FIG. 2B is a schematic representation of the events that are believed tooccur and result in the stable inactivation of the FAS gene inSchizochytrium.

FIG. 3 is a digitized image of the synthesis of fatty acids from[1-¹⁴C]-malonyl-CoA in cell free homogenates from a cell wall deficientstrain of Schizochytrium (AC66) and mutants of that strain in whicheither the PUFA polyketide synthase orfC gene (PUFA-KO) or the FAS gene(FAS-KO) have been inactivated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to a fatty acid synthase (FAS)from Schizochytrium, to biologically active fragments and homologuesthereof, to a nucleic acid sequence encoding such FAS, fragments andhomologues thereof, to the gene encoding Schizochytrium FAS, to hostcells and organisms that recombinantly express the FAS, to host cellsand organisms in which the expression and/or activity of the endogenousFAS has been attenuated, and to various methods for making and using anyof these proteins, nucleic acid molecules, genes, host cells ororganisms. The FAS protein of the present invention is a protein withmultiple enzymatic domains that are homologous, in terms of both aminoacid sequences and in linear domain organization, to the enzymaticdomains encompassed by two proteins in fungi. The FAS domains in mammalsare also found on one large protein, but the organization and thespecific types of enzymatic activities of the domains are significantlydifferent than the Schizochytrium and fungal FAS. Additionally, theamino acid sequence homology between the Schizochytrium FAS domains andthose found in mammalian FAS is significantly less than betweenSchizochytrium and fungal FAS.

This discovery of a multi-functional protein provides a novel approachfor the economic production of short chain fatty acids. For example, itis now possible to clone and express one gene with key sequentialenzymatic functions that are found in at least two genes in other(non-mammalian) organisms, which will greatly facilitate the geneticmodification of production organisms. In addition, it is possible to usethe enzymatic domains of the Schizochytrium FAS gene individually or invarious combinations to construct various recombinant/synthetic genesexpressing, one or more of the domains.

More specifically, the present inventors have cloned a region of genomicDNA from Schizochytrium sp. ATCC 20888 that contains a single orf (openreading frame) that encodes a fatty acid synthase (FAS gene). Theputative function of the enzyme, (i.e., synthesis of short chainsaturated fatty acids such as C14:0 and C16:0), was verified by showingthat strains in which the gene had been disrupted requiresupplementation with short chain fatty acids for survival. The FASencoded by the Schizochytrium gene has some novel features. Theorganization of the domains in the protein is similar to that found inmany fungi (e.g., baker's yeast). However, in all of the fungal enzymescharacterized to date, the FAS is encoded in two subunits (i.e., twoproteins), while the Schizochytrium FAS is one large protein. Based onhomology to the fungal systems, it is likely that the Schizochytrium FASuses acetyl-CoA and malonyl-CoA along with NADH and NADPH as substrates.The product is probably released as an acyl-CoA ester. As in yeast, theprotein contains a phosphopantetheinyl transferase domain that isbelieved to activate an embedded acyl carrier protein domain. TheSchizochytrium FAS is an ‘all-in-one’protein for short chain saturatedfatty acid synthesis.

As used herein, a short chain fatty acid is defined as a fatty acidhaving 18 or fewer carbons. The FAS system of the invention producesshort chain fatty acids, which can include any short chain fatty acid,and typically fatty acids having 16, 14, or 12 carbons, and includesaturated and monounsaturated fatty acids. For example, short chainfatty acids produced by a FAS system include, but are not limited to,short chain saturated fatty acids such as C14:0 and C16:0. The presentinvention encompasses the production of any product of the FAS system.

Schizochytrium is a marine microalga that has been developed as acommercial source of oil enriched in DHA. The DHA in Schizochytrium isthe product of a highly specialized PUFA synthase (described in PCTPublication No. WO 00/42195 and PCT Publication No. WO 02/083870). Herethe present inventors describe the FAS that is responsible for producingthe other major fatty acids (C14:0 and C16:0) found in Schizochytriumoil. The products of the FAS of the present invention are of interest bythemselves and as a major component of the DHA-enriched oil. Alterationof the activity of the FAS in Schizochytrium may influence the relativeamounts of DHA and medium chain saturated fatty acids that accumulate inthat oil, thereby altering its commercial value as a DHA-enriched oil.

Accordingly, the Schizochytrium FAS gene and its encoded product asdescribed herein have several uses. First, subclones of the FAS genomicregion can be used to make a knockout plasmid construct and to createmutants of Schizochytrium in which the FAS gene has been inactivated.Such constructs have already been produced by the inventors and aredescribed herein (see Examples 2 and 3). These mutants may have utilityin a variety of biochemical and genetic studies. In addition,attenuation of the expression and/or activity of the FAS gene inThraustochytrids such as Schizochytrium is a particularly preferredembodiment of the invention, because reduction of FAS activity inThraustochytrids is predicted to increase the accumulation of highlydesirable long chain fatty acids in the organism. For example, oneembodiment of the invention relates to an organism of the order,Thraustochytriales, in which the FAS gene expression is attenuated(resulting in reduced FAS activity), thereby increasing the accumulationof long chain fatty acids and particularly, polyunsaturated fatty acids(PUFAs), by the organism. According to the present invention, referenceto an attenuated gene or protein is to a gene or protein that is notdeleted or completely activated, but for which the expression and/orbiological activity has been reduced (inhibited, down-regulated,decreased) as compared to the expression and/or biological activity ofthe wild-type gene or protein under normal conditions. Therefore, a FAShaving attenuated expression or activity is still expressed and stillhas some biological activity (e.g., so that an organism expressing theFAS is viable), but the expression or biological activity is reduced ascompared to the wild-type FAS.

In another embodiment, expression of this gene in heterologous systems(such as in the cytoplasm of plant cells) is expected to lead to theproduction and accumulation of short chain saturated fatty acids inthose cells. This will provide a means to produce oils enriched in shortchain fatty acids in commercial oil-seed crops. This would be analternative method to the current use of chain-length specificthioesterases targeted to the plastids of plant cells. The end productof the Schizochytrium FAS is likely to be an ester of CoA, and thereforeit would be compatible with oil and phospholipid synthesis in the plantcell cytoplasm.

Accordingly, one embodiment of the present invention relates to anisolated fatty acid synthase (FAS). As used herein, reference to anisolated protein, including an isolated FAS, is to a protein (includinga polypeptide or peptide) that has been removed from its natural milieu(i.e., that has been subject to human manipulation) and can includepurified proteins, partially purified proteins, recombinantly producedproteins, and synthetically produced proteins, for example. As such,“isolated” does not reflect the extent to which the protein has beenpurified. Preferably, an isolated FAS of the present invention isproduced recombinantly. In addition, and by way of example, a“Schizochytrium FAS” refers to a FAS (generally including a homologue ofa naturally occurring FAS) from a Schizochytrium or to a FAS proteinthat has been otherwise produced from the knowledge of the structure(e.g., sequence) and perhaps the function of a naturally occurring FASfrom Schizochytrium. In other words, a Schizochytrium FAS includes anyFAS that has substantially similar structure and function of a naturallyoccurring FAS from Schizochytrium or that is a biologically active(i.e., has biological activity) homologue of a naturally occurring FASfrom Schizochytrium as described in detail herein. As such, aSchizochytrium FAS protein can include purified, partially purified,recombinant, mutated/modified and synthetic proteins. According to thepresent invention, the terms “modification” and “mutation” can be usedinterchangeably, particularly with regard to the modifications/mutationsto the amino acid sequences of FAS (or nucleic acid sequences) describedherein.

According to the present invention, a homologue of a FAS protein (i.e.,a FAS homologue) includes FAS proteins in which at least one or a few,but not limited to one or a few, amino acids have been deleted (e.g., atruncated version of the protein, such as a peptide or fragment),inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, famasylation, amidation and/or addition ofglycosylphosphatidyl inositol). In a preferred embodiment, a FAShomologue has measurable or detectable FAS enzymatic activity (i.e., hasbiological activity). Measurable or detectable FAS enzymatic activitycan include the enzymatic activity of just one, two, three, etc., up toall ten of the functional domains in the FAS protein of the presentinvention (discussed in detail below). In another embodiment, a FAShomologue may or may not have measurable FAS functional (biological)activity, but is used for the preparation of antibodies or thedevelopment of oligonucleotides useful for identifying other FASproteins. For example, the production of an antibody against FAS andproduction of probes and primers useful in the cloning of a FAS areuseful for tracking the presence of FAS nucleic acids or proteins ingenetically modified organisms or for identifying naturally occurringFAS homologues in other organisms (e.g., in other members ofThraustochytriales).

FAS homologues can be the result of natural allelic variation or naturalmutation. FAS homologues of the present invention can also be producedusing techniques known in the art including, but not limited to, directmodifications to the protein or modifications to the gene encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis. A naturally occurring allelicvariant of a nucleic acid encoding a FAS protein is a gene that occursat essentially the same locus (or loci) in the genome as the gene whichencodes the FAS protein of the present invention (e.g., SEQ ID NO:2),but which, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Natural allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art. Homologuescan be produced using techniques known in the art for the production ofproteins including, but not limited to, direct modifications to theisolated, naturally occurring protein, direct protein synthesis, ormodifications to the nucleic acid sequence encoding the protein using,for example, classic or recombinant DNA techniques to effect random ortargeted mutagenesis.

Modifications in FAS homologues, as compared to the wild-type protein,increase, decrease, or do not substantially change, the basic biologicalactivity of the FAS homologue as compared to the naturally occurringprotein, FAS. In general, the biological activity or biological actionof a protein refers to any function(s) exhibited or performed by theprotein that is ascribed to the naturally occurring form of the proteinas measured or observed in vivo (i.e., in the natural physiologicalenvironment of the protein) or in vitro (i.e., under laboratoryconditions). Modifications of a protein, such as in a homologue ormimetic (discussed below), may result in proteins having the samebiological activity as the naturally occurring protein, or in proteinshaving decreased or increased biological activity as compared to thenaturally occurring protein. Modifications which result in a decrease inprotein expression or a decrease in the activity of the protein, can bereferred to as inactivation (complete or partial), down-regulation, ordecreased action of a protein. Similarly, modifications which result inan increase in protein expression or an increase in the activity of theprotein, can be referred to as amplification, overproduction,activation, enhancement, up-regulation or increased action of a protein.

According to one embodiment of the present invention, a biologicallyactive FAS, including a biologically active homologue or fragmentthereof, has at least one characteristic of biological activity ofactivity a wild-type, or naturally occurring, FAS protein describedherein. A FAS biological activity includes the ability to catalyze thesynthesis of short chain fatty acids, including by using acetyl-CoA andmalonyl-CoA along with NADH and NADPH as substrates to produce a shortchain fatty acid. More particularly, a FAS biological activity caninclude any one or more of the biological activities of the nine domainsof FAS described herein. According to the present invention, a FASprotein of the present invention has at least one, and preferably two,and more preferably three, and more preferably four, and more preferablyfive, and more preferably six, and more preferably seven, and morepreferably eight, and most preferably nine, biological activities. Thesebiological activities are: (1) acetyl-transferase (AT) activity; (2)enoyl ACP reductase (ER) activity; (3) dehydratase (DH) activity; (4)malonyl/palmitoyl acyltransferase (M/PAT) activity; (5) a first acylcarrier protein (ACP) activity; (6) a second acyl carrier protein (ACP)activity; (7) keto-acyl ACP reductase (KR) activity; (8) keto-acyl ACPsynthase (KS) activity; and (9) phosphopantetheinyl transferase (PPT)activity. General reference to FAS biological activity typically refersto all biological activities, but does not exclude reference to onlyone, two, three, four, five, six, seven or eight of the biologicalactivities. Methods for measuring these various activities are known inthe art.

Methods to measure protein expression levels according to thisinvention, include, but are not limited to: Western blot, immunoblot,enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),immunoprecipitation, surface plasmon resonance, chemiluminescence,fluorescent polarization, phosphorescence, immunohistochemical analysis,matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry, microcytometry, microarray, microscopy, fluorescenceactivated cell sorting (FACS), and flow cytometry, as well as assaysbased on a property of the protein including but not limited tosubstrate binding. Binding assays are also well known in the art. Forexample, a BIAcore machine can be used to determine the binding constantof a complex between two proteins. The dissociation constant for thecomplex can be determined by monitoring changes in the refractive indexwith respect to time as buffer is passed over the chip (O'Shannessy etal. Anal. Biochem. 212:457-468 (1993); Schuster et al., Nature365:343-347 (1993)). Other suitable assays for measuring the binding ofone protein to another include, for example, immunoassays such as enzymelinked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), ordetermination of binding by monitoring the change in the spectroscopicor optical properties of the proteins through fluorescence, UVabsorption, circular dichrosim, or nuclear magnetic resonance (NMR).

FIG. 1 is a schematic drawing showing the domain organization of theSchizochytrium FAS protein of the present invention as compared to thedomain organization of the individual α and β subunit proteins of yeastFAS systems.

The complete Schizochytrium FAS-encoding sequence is a 12,408 nucleotidesequence (not including the stop codon), represented herein by SEQ IDNO:1, which encodes a 4136 amino acid sequence, represented herein asSEQ ID NO:2. Within the Schizochytrium FAS protein are nine domains asdescribed above: (a) one acetyl-transferase (AT) domain; (b) one enoylACP reductase (ER) domain; (c) one dehydrase (DH) domain; (d) onemalonyl/palmitoyl acyltransferase (M/PAT) domain; (e) two acyl carrierprotein (ACP) domains; (f) one keto-acyl ACP reductase (KR) domain; (g)one keto-acyl ACP synthase (KS) domain; and (h) one phosphopantetheinyltransferase (PPT) domain.

SEQ ID NO:2 was compared with known sequences in a standard proteinBLAST search (BLAST 2.0 Basic BLAST homology search using blastp foramino acid searches, wherein the query sequence is filtered for lowcomplexity regions by default (described in Altschul, S. F., Madden, T.L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety)). The BLAST search used the followingparameters: low complexity filter Off, matrix=BLOSUM62, gap penaltiesare: Existence: 11, Extension 1. BLAST results of entire FAS proteinrevealed that the Schizochytrium FAS can be viewed in general, as aprotein with homology to a head to tail fusion of fungal α and βFASsubunits. All of the domains currently identified in the two yeastproteins have counterparts in the Schizochytrium FAS. In addition, theSchizochytrium FAS has a second ACP domain. In all cases, the bestmatches in the BLAST results are fungi such as: Saccharomycescerevisiae, Candida albicans, Neurospora crassa, Saccharomyces kluyveri,Aspergillus nidulans and Yarrowia lipolytica. At the amino acid level,the sequences with the greatest degree of homology to SEQ ID NO:2 were:(1) the Candida albicans FAS β-subunit (GenBank Accession No. P34731),which was 34% identical to amino acids 1-2100 of SEQ ID NO:2 (identitywas actually over 2038 amino acids of this region of SEQ ID NO:2); and(2) Candida albicans FAS α-subunit (GenBank Accession No. P43098), whichwas 41% identical to amino acids 2101-4136 of SEQ ID NO:2 (identity wasactually over 1864 amino acids of this region of SEQ ID NO:2). Severalother yeast strains showed similar homology to this portion ofSchizochytrium FAS. Since yeast FAS systems have only one ACP domain,whereas the Schizochytrium FAS described herein has two ACP domains, thehomology to the yeast α-subunit is found from the second of the two ACPsin Schizochytrium through the end of the protein.

The first domain in the Schizochytrium FAS protein is anacetyl-transferase (AT) domain, also referred to herein as FAS-AT. Thisdomain is contained within a region of SEQ ID NO:2 spanning from aboutposition 1 to about position 500 of SEQ ID NO:2. The amino acid sequencecontaining the FAS-AT domain is represented herein as SEQ ID NO:5(positions 45-415 of SEQ ID NO:2). BLAST results show that this domainhas high homology to the first domain of several fungal FAS β-subunits.The fungal protein having the closest identity to this domain of the FASprotein of the present invention is a portion of a Yarrowia lipolyticaFAS β-subunit (GenBank Accession No. P34229), which is 30% identicalover 392 amino acids when compared to SEQ ID NO:5 (i.e., containing theAT domain of the FAS of the present invention). An AT generally refersto a class of enzymes that can carry out a number of distinct acyltransfer reactions. The fungal FAS β-subunit has been shown to havespecifically acetyltransacylase activity, which is related to malonylacyltransferase, except that it transfers an acetyl group which servesas the primer for fatty acid synthesis. In the AT domain, the activesite motif is identified as GHS*XG, which in SEQ ID NO:2 is GHS*QG(positions 156-160 of SEQ ID NO:2), where S* (position 158 of SEQ IDNO:2) is the acyl group binding site.

The second domain in the Schizochytrium FAS protein is an enoylreductase (ER) domain, also referred to herein as FAS-ER. This domain iscontained within a region of SEQ ID NO:2 spanning from about position450 to about position 1300 of SEQ ID NO:2. The amino acid sequencecontaining the FAS-ER domain is represented herein as SEQ ID NO:6(positions 460-1210 of SEQ ID NO:2). BLAST results show that this domainhas high homology to the second domain of several fungal FAS β-subunits.The fungal protein having the closest identity to this domain of the FASprotein of the present invention is a portion of a Emericella nidulansFAS β-subunit (GenBank Accession No. AAB41494.1), which is 46% identicalover 561 amino acids when compared to SEQ ID NO:6 (i.e., containing theER domain of the FAS of the present invention). An ER enzyme reduces thetrans-double bond (introduced by the DH activity) in the fatty acyl-ACP,resulting in fully saturating those carbons.

The third domain in the Schizochytrium FAS protein is a dehydratase (DH)domain, also referred to herein as FAS-DH. This domain is containedwithin a region of SEQ ID NO:2 spanning from about position 1300 toabout position 1700 of SEQ ID NO:2. The amino acid sequence containingthe FAS-DH domain is represented herein as SEQ ID NO:7 (positions1450-1575 of SEQ ID NO:2). BLAST results show that this domain has highhomology to the third domain of several fungal FAS β-subunits. Thefungal protein having the closest identity to this domain of the FASprotein of the present invention is the Magnaporthe grisea monoamineoxidase C (MaoC) protein (GenBank Accession No. EAA50359.1), which is39% identical over 121 amino acids when compared to SEQ ID NO:7 (i.e.,containing the DH domain of the FAS of the present invention). MaoCshares similarity with a family of proteins with a variety of functions,including the FAS β-keto-acyl dehydratase (DH) activity. This class ofenzymes removes HOH from a β-keto acyl-ACP and leaves a trans doublebond in the carbon chain.

The fourth domain in the Schizochytrium FAS protein is amalonyl/palmitoyl acyltransferase (M/PAT) domain, also referred toherein as FAS-M/PAT. This domain is contained within a region of SEQ IDNO:2 spanning from about position 1575 to about position 2100 of SEQ IDNO:2. The amino acid sequence containing the FAS-M/PAT domain isrepresented herein as SEQ ID NO:8 (positions 1575-2025 of SEQ ID NO:2).BLAST results show that this domain has high homology to the fourthdomain of several fungal FAS β-subunits. The fungal protein having theclosest identity to this domain of the FAS protein of the presentinvention is a portion of a Neurospora crassa protein (GenBank AccessionNo. EAA33229.1), which is 47% identical over 397 amino acids whencompared to SEQ ID NO:8 (i.e., containing the M/PAT domain of the FAS ofthe present invention). In yeast FAS, FabD (malonyl-CoA:ACPacyltransferase) has been shown to have the dual functions oftransferring the malonyl group from CoA to the FAS ACP domain, and alsoof transferring the fatty acid product of FAS (a palmitoyl group) fromthe FAS ACP to CoA. The active site motif of this protein is GHS*XG,which in the Schizochytrium FAS-M/PAT is GHS*LG (positions 1723-1727 ofSEQ ID NO:2), where the S* (position 1725 of SEQ ID NO:2) is theposition where the acyl group binds.

The fifth and sixth domains in the Schizochytrium FAS protein are acylcarrier protein (ACP) domains, also referred to herein as FAS-ACP. Thesedomains are contained within a region of SEQ ID NO:2 spanning from aboutposition 2025 to about position 2850 of SEQ ID NO:2. The amino acidsequence containing the first FAS-ACP domain (FAS-ACP1) is representedherein as SEQ ID NO:9 (positions 2140-2290 of SEQ ID NO:2). The aminoacid sequence containing the second FAS-ACP domain (FAS-ACP2) isrepresented herein as SEQ ID NO:10 (positions 2325-2585 of SEQ ID NO:2).BLAST results show that these domains have high homology to theN-terminus of several yeast FAS α-subunits which have been designated inthe literature as ACP domains. However, all fungal FAS proteins appearto have only one ACP domain, whereas the Schizochytrium FAS protein ofthe present invention has two ACP domains. The fungal protein having theclosest identity to the first ACP domain of the FAS protein of thepresent invention is a portion of a Neurospora crassa protein (GenBankAccession No. EAA33230.1), which is 44% identical over 149 amino acidswhen compared to SEQ ID NO:9 (i.e., containing the first ACP domain ofthe FAS of the present invention). The fungal protein having the closestidentity to the second ACP domain of the FAS protein of the presentinvention is a portion of a Neurospora crassa protein (GenBank AccessionNo. EAA33230.1), which is 36% identical over 262 amino acids whencompared to SEQ ID NO:10 (i.e., containing the second ACP domain of theFAS of the present invention). By alignment with the yeast sequences,two putative phosphopanthelyation sites are identified in the ACPdomains: the serine residues at positions 2160 (in FAS-ACP1) and 2353(in FAS-ACP-2) of SEQ ID NO:2. A domain or protein having acyl carrierprotein (ACP) biological activity (function) is characterized as being asmall polypeptide (typically, 80 to 100 amino acids long), thatfunctions as a carrier for growing fatty acyl chains via a thioesterlinkage to a covalently bound co-factor of the protein. They occur asseparate units or as domains within larger proteins. ACPs are convertedfrom inactive apo-forms to functional holo-forms by transfer of thephosphopantetheinyl moeity of CoA to a highly conserved serine residueof the ACP. Acyl groups are attached to ACP by a thioester linkage atthe free terminus of the phosphopantetheinyl moiety.

The seventh domain in the Schizochytrium FAS protein is a keto-acyl ACPreductase (KR) domain, also referred to herein as FAS-KR. This domain iscontained within a region of SEQ ID NO:2 spanning from about position2800 to about position 3350 of SEQ ID NO:2. The amino acid sequencecontaining the FAS-KR domain is represented herein as SEQ ID NO:11(positions 2900-3100 of SEQ ID NO:2). BLAST results show that thisdomain has high homology to the second domain of several fungal FASα-subunits (FabG or β-keto-acyl ACP reductase). The fungal proteinhaving the closest identity to this domain of the FAS protein of thepresent invention is a portion of a Neurospora crassa protein (GenBankAccession No. EAA33220.1), which is 50% identical over 205 amino acidswhen compared to SEQ ID NO:11 (i.e., containing the KR domain of the FASof the present invention). As in the yeast FAS, a domain or proteinhaving keto-acyl ACP reductase (KR) biological activity (function), ischaracterized as an enzyme that catalyzes thepyridine-nucleotide-dependent reduction of β-keto acyl forms of ACP. Itis the first reductive step in the de novo fatty acid biosynthesiselongation cycle.

The eighth domain in the Schizochytrium FAS protein is a keto-acyl ACPsynthase (KS) domain, also referred to herein as FAS-KS. This domain iscontained within a region of SEQ ID NO:2 spanning from about position3300 to about position 3900 of SEQ ID NO:2. The amino acid sequencecontaining the FAS-KS domain is represented herein as SEQ ID NO:12(positions 3350-3875 of SEQ ID NO:2). BLAST results show that thisdomain has high homology to the third domain of several fungal FASα-subunits, and particularly, FabB (β-keto-acyl ACP synthase). Thefungal protein having the closest identity to this domain of the FASprotein of the present invention is a portion of a Candida albicans FASα-subunit protein (GenBank Accession No. P43098), which is 55% identicalover 548 amino acids when compared to SEQ ID NO:12 (i.e., containing theKS domain of the FAS of the present invention). The active site cysteineof this domain has been identified at position 3530 of SEQ ID NO:2.According to the present invention, a domain or protein having (KS)biological activity (function) is characterized as an enzyme thatcarries out the initial step of the FAS elongation reaction cycle. Theacyl group destined for elongation is linked to a cysteine residue atthe active site of the enzyme by a thioester bond. In the multi-stepreaction, the acyl-enzyme undergoes condensation with malonyl-ACP toform -keto-acyl-ACP, CO₂ and free enzyme. The KS plays a key role in theelongation cycle and in many systems has been shown to possess greatersubstrate specificity than other enzymes of the reaction cycle.

The ninth domain in the Schizochytrium FAS protein is aphosphopantetheinyl transferase (PPT domain), also referred to herein asFAS-PPT. This domain is contained within a region of SEQ ID NO:2spanning from about position 3900 to about position 4136 of SEQ ID NO:2.The amino acid sequence containing the FAS-PPT domain is representedherein as SEQ ID NO:13 (positions 4025-4136 of SEQ ID NO:2). BLASTresults show that this domain has high homology to the C-terminal domainof several fungal FAS α-subunits, and particularly, to AcpS (holo-ACPsynthase, also known as 4-phosphopantetheinyl transferase). The fungalprotein having the closest identity to this domain of the FAS protein ofthe present invention is a portion of a Schizosaccharomyces pombe FASα-subunit protein (GenBank Accession No. BAB62031.1), which is 46%identical over 110 amino acids when compared to SEQ ID NO:13 (i.e.,containing the PPT domain of the FAS of the present invention). A PPTdomain is required for the attachment of a phosphopantetheine cofactorto produce the active, holo-ACP.

In one embodiment, a FAS protein (e.g., including homologues of the FASisolated from Schizochytrium and described in detail herein) includesproteins that have at least one of: (a) acetyl-transferase (AT)activity; (b) enoyl ACP reductase (ER) activity; (c) dehydratase (DH)activity; (d) malonyl/palmitoyl acyltransferase (M/PAT) activity; (e)acyl carrier protein (ACP) activity; (f) keto-acyl ACP reductase (KR)activity; (g) keto-acyl ACP synthase (KS) activity; and (h)phosphopantetheinyl transferase (PPT) activity. In one embodiment of theinvention, an isolated FAS comprises an amino acid sequence selectedfrom: (a) an amino acid sequence selected from: SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequenceconsisting of positions 1-500 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequenceconsisting of positions 3900-4136 of SEQ ID NO:2, or biologically activefragments of any of these sequences or any combinations of thesesequences; or, (b) an amino acid sequence that is at least about 45%identical to any of these sequences, wherein the amino acid sequence hasthe biological activity of the reference sequence (biological activitiesof these sequences are described above).

In one aspect of the invention, as discussed above a FAS proteincomprises an amino acid sequence that is at least about 45% identical toany of the above-described amino acid sequences representing thefull-length FAS of the invention (i.e., SEQ ID NO:2), a regioncontaining a biologically active domain of the FAS of the invention(i.e., a region of SEQ ID NO:2 defined by positions numbers above), or abiologically active domain of the FAS of the invention (i.e., any one ofSEQ ID NOs:5-13), over the full length of that protein, region, ordomain. In another aspect, a FAS protein of the invention comprises anamino acid sequence that is at least 50% identical to any of theabove-identified protein, regions or domains, and in another aspect atleast about 55%, and in another aspect at least about 60%, and inanother aspect at least about 65%, and in another aspect at least about70%, and in another aspect at least about 75%, and in another aspect atleast about 80%, and in another aspect at least about 85%, and inanother aspect at least about 90%, and in another aspect at least about95% identical, and in another aspect at least about 96% identical, andin another aspect at least about 97% identical, and in another aspect atleast about 98% identical, and in another aspect at least about 99%identical, to the amino acid sequence defining any of theabove-identified protein, regions or domains. Preferably, a FAS proteinof the present invention comprises at least one, two, three, four, five,six, seven, eight, or all nine biological activities of a FAS protein ofthe invention selected from: (a) acetyl-transferase (AT) activity; (b)enoyl ACP reductase (ER) activity; (c) dehydratase (DH) activity; (d)malonyl/palmitoyl acyltransferase (M/PAT) activity; (e) acyl carrierprotein (ACP) activity; (f) keto-acyl ACP reductase (KR) activity; (g)keto-acyl ACP synthase (KS) activity; and (h) phosphopantetheinyltransferase (PPT) activity.

In one embodiment of the present invention, a FAS homologue according tothe present invention has an amino acid sequence that is less than about100% identical to any of the above-identified amino acid sequences for afull-length FAS protein, or a region or domain thereof according to thepresent invention. In another aspect of the invention, a FAS homologueaccording to the present invention has an amino acid sequence that isless than about 99% identical to any of the above-identified amino acidsequences, and in another embodiment, is less than is less than 98%identical to any of the above-identified amino acid sequences, and inanother embodiment, is less than 97% identical to any of theabove-identified amino acid sequences, and in another embodiment, isless than 96% identical to any of the above-identified amino acidsequences, and in another embodiment, is less than 95% identical to anyof the above-identified amino acid sequences, and in another embodiment,is less than 94% identical to any of the above-identified amino acidsequences, and in another embodiment, is less than 93% identical to anyof the above-identified amino acid sequences, and in another embodiment,is less than 92% identical to any of the above-identified amino acidsequences, and in another embodiment, is less than 91% identical to anyof the above-identified amino acid sequences, and in another embodiment,is less than 90% identical to any of the above-identified amino acidsequences, and so on, in increments of whole integers.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

A FAS protein can also include proteins having an amino acid sequencecomprising at least 10 contiguous amino acid residues of any of theabove-identified amino acid sequences (i.e., 10 contiguous amino acidresidues having 100% identity with 10 contiguous amino acids of thereference amino acid sequence). In another aspect, a homologue of a FASamino acid sequence includes amino acid sequences comprising at least20, or at least about 30, or at least about 40, or at least about 50, orat least about 75, or at least about 100, or at least about 115, or atleast about 130, or at least about 150, or at least about 200, or atleast about 250, or at least about 300, or at least about 350, or atleast about 400, or at least about 500, or at least about 600, or atleast about 700, or at least about 800, or at least about 900, or atleast about 1000, or at least about 1100, or at least about 1200, and soon, in increments of 10 amino acids, up to at least about 4130contiguous amino acid residues of the amino acid sequence represented bySEQ ID NO:2. A FAS homologue can include proteins encoded by a nucleicacid sequence comprising at least about 30, or at least about 60, or atleast about 90, or at least about 150, or at least about 225, or atleast about 300, or at least about 750, or at least about 900, or atleast about 1050, or at least about 1200, or at least about 1500, or atleast about 1800, or at least about 2100, or at least about 2400, or atleast about 2700, or at least about 3000, and so on, in increments of 30nucleotides, up to at least about 12,400 contiguous nucleotides of thenucleic acid sequence represented by SEQ ID NO:1. In a preferredembodiment, a FAS homologue has measurable FAS biological activity(i.e., has biological activity), as described above, including any oneor more of the biological activities described for a FAS of the presentinvention.

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

In another embodiment, a FAS protein, including a FAS homologue,includes a protein having an amino acid sequence that is sufficientlysimilar to a natural FAS amino acid sequence that a nucleic acidsequence encoding the homologue is capable of hybridizing undermoderate, high or very high stringency conditions (described below) to(i.e., with) a nucleic acid molecule encoding the natural FAS (i.e., tothe complement of the nucleic acid strand encoding the natural FAS aminoacid sequence). Preferably, a homologue of a FAS protein is encoded by anucleic acid molecule comprising a nucleic acid sequence that hybridizesunder moderate, high or very high stringency conditions to thecomplement of a nucleic acid sequence that encodes a protein comprisingSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13. Evenmore preferably, a homologue of a FAS protein is encoded by a nucleicacid molecule comprising a nucleic acid sequence that hybridizes undermoderate, high or very high stringency conditions to the complement ofthe nucleic acid sequence represented by SEQ ID NO:1.

A nucleic acid sequence complement of nucleic acid sequence encoding aFAS of the present invention refers to the nucleic acid sequence of thenucleic acid strand that is complementary to the strand which encodesFAS. It will be appreciated that a double stranded DNA which encodes agiven amino acid sequence comprises a single strand DNA and itscomplementary strand having a sequence that is a complement to thesingle strand DNA. As such, nucleic acid molecules of the presentinvention can be either double-stranded or single-stranded, and includethose nucleic acid molecules that form stable hybrids under stringenthybridization conditions with a nucleic acid sequence that encodes theamino acid sequence of SEQ ID NO:2, for example, and/or with thecomplement of the nucleic acid sequence that encodes an amino acidsequence of SEQ ID NO:2. Methods to deduce a complementary sequence areknown to those skilled in the art. It should be noted that since nucleicacid sequencing technologies are not entirely error-free, the sequencespresented herein, at best, represent apparent sequences of a FAS proteinof the present invention.

As used herein, reference to hybridization conditions refers to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

FAS proteins also include expression products of fusions (e.g., fusionproteins, for example, used to overexpress soluble, active forms of therecombinant protein), of mutagenized genes (such as genes having codonmodifications to enhance gene transcription and translation), and oftruncated genes (such as genes having membrane binding domains removedto generate soluble forms of a membrane protein, or genes having signalsequences removed which are poorly tolerated in a particular recombinanthost). It is noted that FAS and protein homologues of the presentinvention include proteins which do not have any FAS activity or morepreferably, that have attenuated FAS activity. Such proteins are useful,for example, for the production of antibodies or for production ofgenetically modified organisms that lack the ability to produce one ormore short chain fatty acids.

The minimum size of a protein and/or homologue of the present inventionis a size sufficient to have FAS biological activity or, when theprotein is not required to have such activity, sufficient to be usefulfor another purpose associated with a FAS protein of the presentinvention, such as for the production of antibodies that bind to anaturally occurring FAS. As such, the minimum size of a FAS or homologueof the present invention is a size suitable to form at least one epitopethat can be recognized by an antibody, and is typically at least 8 aminoacids in length, and preferably 10, and more preferably 15, and morepreferably 20, and more preferably 25, and even more preferably 30 aminoacids in length, and up to 4136 amino acids in length, in increments ofany whole integer from 1 to 4136, with preferred sizes depending onwhether full-length, multivalent (i.e., fusion protein having more thanone domain, each of which has a function), or functional portions ofsuch proteins are desired. There is no limit, other than a practicallimit, on the maximum size of such a protein in that the protein caninclude a portion of a FAS protein (including FAS homologues) or afull-length FAS.

Similarly, the minimum size of a nucleic acid molecule of the presentinvention is a size sufficient to encode a protein having FAS activity(including the activity of one or more domains of a FAS of the presentinvention), sufficient to encode a protein comprising at least oneepitope which binds to an antibody, or sufficient to form a probe oroligonucleotide primer that is capable of forming a stable hybrid withthe complementary sequence of a nucleic acid molecule encoding a naturalFAS (e.g., under low, moderate or high stringency conditions). As such,the size of the nucleic acid molecule encoding such a protein can bedependent on nucleic acid composition and percent homology or identitybetween the nucleic acid molecule and complementary sequence as well asupon hybridization conditions per se (e.g., temperature, saltconcentration, and formamide concentration). The minimal size of anucleic acid molecule that is used as an oligonucleotide primer or as aprobe is typically at least about 12 to about 15 nucleotides in lengthif the nucleic acid molecules are GC-rich and at least about 15 to about18 bases in length if they are AT-rich. There is no limit, other than apractical limit, on the maximal size of a nucleic acid molecule of thepresent invention, in that the nucleic acid molecule can include aportion of a FAS encoding sequence, a nucleic acid sequence encoding afull-length FAS (including a FAS gene), or multiple genes, or portionsthereof.

The present invention also includes a fusion protein that includes aFAS-containing domain (including a homologue or functional domain of aFAS) attached to one or more fusion segments. Suitable fusion segmentsfor use with the present invention include, but are not limited to,segments that can: enhance a protein's stability; provide otherdesirable biological activity; and/or assist with the purification of aFAS protein (e.g., by affinity chromatography). A suitable fusionsegment can be a domain of any size that has the desired function (e.g.,imparts increased stability, solubility, action or biological activity;and/or simplifies purification of a protein). Fusion segments can bejoined to amino and/or carboxyl termini of the FAS-containing domain ofthe protein and can be susceptible to cleavage in order to enablestraight-forward recovery of a FAS protein. Fusion proteins arepreferably produced by culturing a recombinant cell transformed with afusion nucleic acid molecule that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of aFAS-containing domain.

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” the specified amino acid sequence. According to thepresent invention, the heterologous amino acids are a sequence of aminoacids that are not naturally found (i.e., not found in nature, in vivo)flanking the specified amino acid sequence, or that are not related tothe function of the specified amino acid sequence, or that would not beencoded by the nucleotides that flank the naturally occurring nucleicacid sequence encoding the specified amino acid sequence as it occurs inthe gene, if such nucleotides in the naturally occurring sequence weretranslated using standard codon usage for the organism from which thegiven amino acid sequence is derived. Similarly, the phrase “consistingessentially of”, when used with reference to a nucleic acid sequenceherein, refers to a nucleic acid sequence encoding a specified aminoacid sequence that can be flanked by from at least one, and up to asmany as about 60, additional heterologous nucleotides at each of the 5′and/or the 3′ end of the nucleic acid sequence encoding the specifiedamino acid sequence. The heterologous nucleotides are not naturallyfound (i.e., not found in nature, in vivo) flanking the nucleic acidsequence encoding the specified amino acid sequence as it occurs in thenatural gene or do not encode a protein that imparts any additionalfunction to the protein or changes the function of the protein havingthe specified amino acid sequence.

FAS proteins as described herein can be isolated from a variousmicroorganisms including members of the order Thraustochytriales. Forexample, preferred microorganisms from which a FAS protein of thepresent invention may be derived include microorganisms from a genusincluding, but not limited to: Thraustochytrium, Labyrinthuloides,Japonochytrium, and Schizochytrium. Preferred species within thesegenera include, but are not limited to: any Schizochytrium species,including Schizochytrium aggregatum, Schizochytrium limacinum,Schizochytrium minutum; any Thraustochytrium species (including formerUlkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U.profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and includingThraustochytrium striatum, Thraustochytrium aureum, Thraustochytriumroseum; and any Japonochytrium species. Particularly preferred strainsof Thraustochytriales include, but are not limited to: Schizochytriumsp. (S31)(ATCC 20888); Schizochytrium sp. (S8)(ATCC 20889);Schizochytrium sp. (LC-RM)(ATCC 18915); Schizochytrium sp. (SR21);Schizochytrium aggregatum (Goldstein et Belsky)(ATCC 28209);Schizochytrium limacinum (Honda et Yokochi)(IFO 32693); Thraustochytriumsp. (23B)(ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC24473); Thraustochytrium aureum (Goldstein)(ATCC 34304);Thraustochytrium roseum (Goldstein)(ATCC 28210); and Japonochytrium sp.(L1)(ATCC 28207).

According to the present invention, the terms/phrases “Thraustochytrid”,“Thraustochytriales microorganism” and “microorganism of the orderThraustochytriales” can be used interchangeably and refer to any membersof the order Thraustochytriales, which includes both the familyThraustochytriaceae and the family Labyrinthulaceae. The terms“Labyrinthulid” and “Labyrinthulaceae” are used herein to specificallyrefer to members of the family Labyrinthulaceae. To specificallyreference Thraustochytrids that are members of the familyThraustochytriaceae, the term “Thraustochytriaceae” is used herein.Thus, for the present invention, members of the Labyrinthulids areconsidered to be included in the Thraustochytrids.

Developments have resulted in frequent revision of the taxonomy of theThraustochytrids. Taxonomic theorists generally place Thraustochytridswith the algae or algae-like protists. However, because of taxonomicuncertainty, it would be best for the purposes of the present inventionto consider the strains described in the present invention asThraustochytrids to include the following organisms: Order:Thraustochytriales; Family: Thraustochytriaceae (Genera:Thraustochytrium, Schizochytrium, Japonochytrium, Aplanochytrium, orElina) or Labyrinthulaceae (Genera Labyrinthula, Labyrinthuloides, orLabyrinthomyxa). Also, the following genera are sometimes included ineither family Thraustochytriaceae or Labyrinthulaceae: Althornia,Corallochytrium, Diplophyrys, and Pyrrhosorus), and for the purposes ofthis invention are encompassed by reference to a Thraustochytrid or amember of the order Thraustochytriales. It is recognized that at thetime of this invention, revision in the taxonomy of Thraustochytridsplaces the genus Labyrinthuloides in the family of Labyrinthulaceae andconfirms the placement of the two families Thraustochytriaceae andLabyrinthulaceae within the Stramenopile lineage. It is noted that theLabyrinthulaceae are sometimes commonly called labyrinthulids orlabyrinthula, or labyrinthuloides and the Thraustochytriaceae arecommonly called thraustochytrids, although, as discussed above, for thepurposes of clarity of this invention, reference to Thraustochytridsencompasses any member of the order Thraustochytriales and/or includesmembers of both Thraustochytriaceae and Labyrinthulaceae. Recenttaxonomic changes are summarized below.

Strains of certain unicellular microorganisms disclosed herein aremembers of the order Thraustochytriales. Thraustochytrids are marineeukaryotes with an evolving taxonomic history. Problems with thetaxonomic placement of the Thraustochytrids have been reviewed by Moss(in “The Biology of Marine Fungi”, Cambridge University Press p. 105(1986)), Bahnweb and Jackle (ibid. p. 131) and Chamberlain and Moss(BioSystems 21:341 (1988)).

For convenience purposes, the Thraustochytrids were first placed bytaxonomists with other colorless zoosporic eukaryotes in thePhycomycetes (algae-like fungi). The name Phycomycetes, however, waseventually dropped from taxonomic status, and the Thraustochytrids wereretained in the Oomycetes (the biflagellate zoosporic fungi). It wasinitially assumed that the Oomycetes were related to the heterokontalgae, and eventually a wide range of ultrastructural and biochemicalstudies, summarized by Barr (Barr. Biosystems 14:359 (1981)) supportedthis assumption. The Oomycetes were in fact accepted by Leedale(Leedale. Taxon 23:261 (1974)) and other phycologists as part of theheterokont algae. However, as a matter of convenience resulting fromtheir heterotrophic nature, the Oomycetes and Thraustochytrids have beenlargely studied by mycologists (scientists who study fungi) rather thanphycologists (scientists who study algae).

From another taxonomic perspective, evolutionary biologists havedeveloped two general schools of thought as to how eukaryotes evolved.One theory proposes an exogenous origin of membrane-bound organellesthrough a series of endosymbioses (Margulis, 1970, Origin of EukaryoticCells. Yale University Press, New Haven); e.g., mitochondria werederived from bacterial endosymbionts, chloroplasts from cyanophytes, andflagella from spirochaetes. The other theory suggests a gradualevolution of the membrane-bound organelles from the non-membrane-boundedsystems of the prokaryote ancestor via an autogenous process(Cavalier-Smith, 1975, Nature (Lond.) 256:462-468). Both groups ofevolutionary biologists however, have removed the Oomycetes andThraustochytrids from the fungi and place them either with thechromophyte algae in the kingdom Chromophyta (Cavalier-Smith BioSystems14:461 (1981)) (this kingdom has been more recently expanded to includeother protists and members of this kingdom are now called Stramenopiles)or with all algae in the kingdom Protoctista (Margulis and Sagen.Biosystems 18:141 (1985)).

With the development of electron microscopy, studies on theultrastructure of the zoospores of two genera of Thraustochytrids,Thraustochytrium and Schizochytrium, (Perkins, 1976, pp. 279-312 in“Recent Advances in Aquatic Mycology” (ed. E. B. G. Jones), John Wiley &Sons, New York; Kazama. Can. J. Bot. 58:2434 (1980); Barr, 1981,Biosystems 14:359-370) have provided good evidence that theThraustochytriaceae are only distantly related to the Oomycetes.Additionally, genetic data representing a correspondence analysis (aform of multivariate statistics) of 5-S ribosomal RNA sequences indicatethat Thraustochytriales are clearly a unique group of eukaryotes,completely separate from the fungi, and most closely related to the redand brown algae, and to members of the Oomycetes (Mannella et al. Mol.Evol. 24:228 (1987)). Most taxonomists have agreed to remove theThraustochytrids from the Oomycetes (Bartnicki-Garcia. p. 389 in“Evolutionary Biology of the Fungi” (eds. Rayner, A. D. M., Brasier, C.M. & Moore, D.), Cambridge University Press, Cambridge).

In summary, employing the taxonomic system of Cavalier-Smith(Cavalier-Smith. BioSystems 14:461 (1981); Cavalier-Smith. Microbiol.Rev. 57:953 (1993)), the Thraustochytrids are classified with thechromophyte algae in the kingdom Chromophyta (Stramenopiles). Thistaxonomic placement has been more recently reaffirmed by Cavalier-Smithet al. using the 18s rRNA signatures of the Heterokonta to demonstratethat Thraustochytrids are chromists not Fungi (Cavalier-Smith et al.Phil. Tran. Roy. Soc. London Series BioSciences 346:387 (1994)). Thisplaces the Thraustochytrids in a completely different kingdom from thefungi, which are all placed in the kingdom Eufungi.

Currently, there are 71 distinct groups of eukaryotic organisms(Patterson. Am. Nat. 154:S96 (1999)) and within these groups four majorlineages have been identified with some confidence: (1) Alveolates, (2)Stramenopiles, (3) a Land Plant-green algae-Rhodophyte_Glaucophyte(“plant”) lade and (4) an Opisthokont lade (Fungi and Animals). Formerlythese four major lineages would have been labeled Kingdoms but use ofthe “kingdom” concept is no longer considered useful by someresearchers.

As noted by Armstrong, Stramenopile refers to three-parted tubularhairs, and most members of this lineage have flagella bearing suchhairs. Motile cells of the Stramenopiles (unicellular organisms, sperm,zoospores) are asymmetrical having two laterally inserted flagella, onelong, bearing three-parted tubular hairs that reverse the thrust of theflagellum, and one short and smooth. Formerly, when the group was lessbroad, the Stramenopiles were called Kingdom Chromista or the heterokont(=different flagella) algae because those groups consisted of the BrownAlgae or Phaeophytes, along with the yellow-green Algae, Golden-brownAlgae, Eustigmatophytes and Diatoms. Subsequently some heterotrophic,fungal-like organisms, the water molds, and labyrinthulids (slime netamoebas), were found to possess similar motile cells, so a group namereferring to photosynthetic pigments or algae became inappropriate.Currently, two of the families within the Stramenopile lineage are theLabyrinthulaceae and the Thraustochytriaceae. Historically, there havebeen numerous classification strategies for these unique microorganismsand they are often classified under the same order (i.e.,Thraustochytriales). Relationships of the members in these groups arestill developing. Porter and Leander have developed data based on 18Ssmall subunit ribosomal DNA indicating the thraustochytrid-labyrinthulidlade in monophyletic. However, the lade is supported by two branches;the first contains three species of Thraustochytrium and Ulkeniaprofunda, and the second includes three species of Labyrinthula, twospecies of Labyrinthuloides and Schizochytrium aggregatum.

The taxonomic placement of the Thraustochytrids as used in the presentinvention is therefore summarized below:

Kingdom: Chromophyta (Stramenopiles)

Phylum: Heterokonta

Order: Thraustochytriales (Thraustochytrids)

Family: Thraustochytriaceae or Labyrinthulaceae

Genera: Thraustochytrium, Schizochytrium, Japonochytrium,Aplanochytrium, Elina, Labyrinthula, Labyrinthuloides, orLabyrinthulomyxa

Some early taxonomists separated a few original members of the genusThraustochytrium (those with an amoeboid life stage) into a separategenus called Ulkenia. However it is now known that most, if not all,Thraustochytrids (including Thraustochytrium and Schizochytrium),exhibit amoeboid stages and as such, Ulkenia is not considered by someto be a valid genus. As used herein, the genus Thraustochytrium willinclude Ulkenia.

Despite the uncertainty of taxonomic placement within higherclassifications of Phylum and Kingdom, the Thraustochytrids remain adistinctive and characteristic grouping whose members remainclassifiable within the order Thraustochytriales.

Further embodiments of the present invention include nucleic acidmolecules that encode a FAS protein. An isolated nucleic acid moleculeof the present invention includes a nucleic acid molecule comprising anucleic acid sequence encoding any of the isolated FAS proteins,including a FAS homologue or fragment, described above.

In one embodiment, such nucleic acid molecules include isolated nucleicacid molecules that hybridize under moderate stringency conditions, andeven more preferably under high stringency conditions, and even morepreferably under very high stringency conditions with the complement ofa nucleic acid sequence encoding a naturally occurring FAS protein(i.e., including naturally occurring allelic variants encoding a FASprotein). Preferably, an isolated nucleic acid molecule encoding a FASprotein of the present invention comprises a nucleic acid sequence thathybridizes under moderate, high, or very high stringency conditions tothe complement of a nucleic acid sequence that encodes any of theproteins described above. In one embodiment, an isolated nucleic acidmolecule comprises a nucleic acid sequence that hybridizes undermoderate, high or very high stringency conditions to the complement of anucleic acid sequence represented by SEQ ID NO:1. Such conditions havebeen described in detail above.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA, includingcDNA. As such, “isolated” does not reflect the extent to which thenucleic acid molecule has been purified. An isolated FAS nucleic acidmolecule of the present invention can be isolated from its naturalsource or produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning) or chemical synthesis.Isolated FAS nucleic acid molecules can include, for example, FAS genes,natural allelic variants of FAS genes, FAS coding regions or portionsthereof, and FAS coding and/or regulatory regions modified by nucleotideinsertions, deletions, substitutions, and/or inversions in a manner suchthat the modifications do not substantially interfere with the nucleicacid molecule's ability to encode a FAS protein of the present inventionor to form stable hybrids under stringent conditions with natural geneisolates. An isolated FAS nucleic acid molecule can includedegeneracies. As used herein, nucleotide degeneracies refers to thephenomenon that one amino acid can be encoded by different nucleotidecodons. Thus, the nucleic acid sequence of a nucleic acid molecule thatencodes a FAS protein of the present invention can vary due todegeneracies. It is noted that an isolated FAS nucleic acid molecule ofthe present invention is not required to encode a protein having FASactivity. A FAS nucleic acid molecule can encode a truncated, mutated orinactive protein, for example. Such nucleic acid molecules and theproteins encoded by such nucleic acid molecules are useful in as probesand primers for the identification of other FAS proteins.

According to the present invention, reference to a FAS gene includes allnucleic acid sequences related to a natural (i.e. wild-type) FAS gene,such as regulatory regions that control production of the FAS encoded bythat gene (such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself.The present inventors provide herein the 5′ untranslated region of theFAS gene of the present invention (represented herein by SEQ ID NO:4),as well as the 3′ untranslated region (represented herein by SEQ IDNO:3). The use of these regions of the FAS gene to regulate theexpression and/or biological activity of a FAS-encoding nucleic acidmolecule, such as an endogenous FAS gene, or to regulate the expressionor activity of a heterologous nucleic acid molecule (i.e., a moleculeencoding a different protein) is encompassed by the present invention.In addition, the regulation of the expression or activity of a FAS geneor protein according to the present invention also encompasses the useof any regulatory sequence or protein, including heterologous regulatorysequences and proteins (i.e., those that are not naturally associatedwith the FAS gene or protein) to regulate the expression and/orbiological activity of FAS. Such uses will be described below.

In another embodiment, a FAS gene can be a naturally occurring allelicvariant that includes a similar but not identical sequence to thenucleic acid sequence encoding a given FAS protein. Allelic variantshave been previously described above. The phrases “nucleic acidmolecule” and “gene” can be used interchangeably when the nucleic acidmolecule comprises a gene as described above.

A FAS nucleic acid molecule homologue (i.e., encoding a FAS homologue)can be produced using a number of methods known to those skilled in theart (see, for example, Sambrook et al.). For example, nucleic acidmolecules can be modified using a variety of techniques including, butnot limited to, by classic mutagenesis and recombinant DNA techniques(e.g., site-directed mutagenesis, chemical treatment, restriction enzymecleavage, ligation of nucleic acid fragments and/or PCR amplification),or synthesis of oligonucleotide mixtures and ligation of mixture groupsto “build” a mixture of nucleic acid molecules and combinations thereof.Another method for modifying a recombinant nucleic acid moleculeencoding a FAS is gene shuffling (i.e., molecular breeding) (See, forexample, U.S. Pat. No. 5,605,793 to Stemmer; Minshull and Stemmer; 1999,Curr. Opin. Chem. Biol. 3:284-290; Stemmer, 1994, P.N.A.S. USA91:10747-10751, all of which are incorporated herein by reference intheir entirety). This technique can be used to efficiently introducemultiple simultaneous changes in the FAS activity. Nucleic acid moleculehomologues can be selected by hybridization with a FAS gene or byscreening the function of a protein encoded by a nucleic acid molecule(e.g., enzymatic activity).

One embodiment of the present invention relates to an oligonucleotide,comprising at least 12 contiguous nucleotides of SEQ ID NO:1, and anucleic acid sequence fully complementary thereto. The minimal size of anucleic acid molecule that is used as an oligonucleotide primer or as aprobe is typically at least about 12 to about 15 nucleotides in lengthif the nucleic acid molecules are GC-rich and at least about 15 to about18 bases in length if they are AT-rich. There is no limit, other than apractical limit, on the maximal size of an oligonucleotide probe orprimer of the present invention, in that the probe or primer can includeany portion of a FAS gene of the invention that is suitable for theintended use, with probes typically being larger than primers. As such,an oligonucleotide of the invention can include any length fragmentbetween about 12 and about 12,408 nucleotides or even larger probes, inwhole integers (e.g., 12, 13, 14, 15, 16 . . . 12,407, 12,408).

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes at least one isolated nucleic acidmolecule of the present invention inserted into any nucleic acid vector(e.g., a recombinant vector) which is suitable for cloning, sequencing,and/or otherwise manipulating the nucleic acid molecule, such asexpressing and/or delivering the nucleic acid molecule into a host cellto form a recombinant cell. Such a vector contains heterologous nucleicacid sequences, that is nucleic acid sequences that are not naturallyfound adjacent to nucleic acid molecules of the present invention,although the vector can also contain regulatory nucleic acid sequences(e.g., promoters, untranslated regions) which are naturally foundadjacent to nucleic acid molecules of the present invention (discussedin detail below). The vector can be either RNA or DNA, eitherprokaryotic or eukaryotic, and typically is a virus or a plasmid. Thevector can be maintained as an extrachromosomal element (e.g., aplasmid) or it can be integrated into the chromosome. The entire vectorcan remain in place within a host cell, or under certain conditions, theplasmid DNA can be deleted, leaving behind the nucleic acid molecule ofthe present invention. The integrated nucleic acid molecule can be underchromosomal promoter control, under native or plasmid promoter control,or under a combination of several promoter controls. Single or multiplecopies of the nucleic acid molecule can be integrated into thechromosome. The vector can be designed for tissue-specific expression inthe host cell, such as by using tissue-specific promoters. Severalrecombinant nucleic acid molecules useful in the present invention,including several recombinant vectors, are described in detail in theExamples.

Typically, a recombinant molecule includes a nucleic acid molecule ofthe present invention operatively linked to one or more transcriptioncontrol sequences (e.g., promoters, operators, repressors, enhancers,terminators). As used herein, the phrase “recombinant molecule” or“recombinant nucleic acid molecule” primarily refers to a nucleic acidmolecule or nucleic acid sequence operatively linked to a transcriptioncontrol sequence, but can be used interchangeably with the phrase“nucleic acid molecule”, when such nucleic acid molecule is arecombinant molecule as discussed herein. According to the presentinvention, the phrase “operatively linked” refers to linking a nucleicacid molecule to a transcription control sequence in a manner such thatthe molecule is able to be expressed when transformed (i.e.,transformed, transduced, transfected, or conjugated) into a host cell.Transcription control sequences are sequences which control theinitiation, elongation, or termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells useful for expressing a FAS protein of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in Thraustochytriales microorganisms,bacterial, fungal (e.g., yeast), or plant cells. Other preferredtranscription control sequences are for plants and include those thatpromote gene expression in specific tissues (e.g., leaves, stems, roots,flowers, seeds) and can be referred to herein as tissue-specifictranscription control sequences. Such sequences are well known in theart.

In one embodiment of the invention, a suitable transcription controlsequence includes the regulatory sequences that are naturally found inthe FAS gene of the present invention. For example, regulatory sequencesof a Schizochytrium FAS, which include a FAS promoter, are found innucleotides represented herein by SEQ ID NO:3 (3′ untranslated region)or in nucleotides represented herein by SEQ ID NO:4 (5′ untranslatedregion). In another embodiment, any regulatory sequence can be usedwhich increases (enhances, upregulates), allows/maintains or decreases(attenuates, downregulates, reduces) the expression and/or biologicalactivity of an endogenous and/or recombinant FAS gene or protein of thepresent invention. Regulatory sequences, including promoter sequences,for a variety of host organisms are known in the art and all areencompassed for use in the present invention.

Recombinant molecules of the present invention, which can be either DNAor RNA, can also contain additional regulatory sequences, such astranscription regulatory sequences, translation regulatory sequences,origins of replication, and other regulatory sequences that arecompatible with the recombinant cell. In one embodiment, a recombinantmolecule of the present invention, including those which are integratedinto the host cell chromosome, also contains signal (targeting) (i.e.,signal segment nucleic acid sequences) to enable an expressed FAS to besecreted from the cell that produces the protein or targeted to aparticular organelle or membrane. For example, in one embodiment,suitable signal segments include a signal segment that is naturallyassociated with a FAS of the present invention or any heterologoussignal segment capable of directing the secretion of a FAS proteinaccording to the present invention. In another embodiment, a recombinantmolecule of the present invention comprises a signal sequence to enablean expressed FAS protein to be delivered to and inserted into themembrane of a host cell. In another embodiment, a recombinant moleculeof the present invention comprises a signal sequence which specificallytargets the delivery of a FAS to specific sub-cellular organelles orcompartments, such as the endoplasmic reticulum, the chloroplast, thechromoplast, other plastids, or the cytoplasm.

One or more recombinant molecules of the present invention can be usedto produce an encoded product (e.g., a FAS protein) of the presentinvention. In one embodiment, an encoded product is produced byexpressing a nucleic acid molecule as described herein under conditionseffective to produce the protein. A preferred method to produce anencoded protein is by transforming a host cell with one or morerecombinant molecules to form a recombinant cell. Suitable host cells totransform include, but are not limited to, any microalgal cell,including a Thraustochytriales microorganism, or any bacterial cell,fungal (e.g., yeast) cell, other microbial cell, or plant cell that canbe transformed. Host cells can be either untransformed cells or cellsthat are already transformed with at least one nucleic acid molecule.

Preferred host cells for use in the present invention include anymicroorganism cell or plant cell which is suitable for expression of aFAS protein of the present invention, including, but not limited to: (1)plants, including, but not limited to, crop plants (e.g.,canola—Brassica napus, rice, corn, flax, safflower, soy, sunflower,rapeseed, linseed); (2) fungi, including, but not limited to, Phycomycessp., Neurospora sp., Mucor sp. (e.g., Mucor circinelloides), Blakesleasp., Mortierella sp. (e.g., Mortierella alpina), Rhodotorula sp.,Lipomyces sp. (e.g., Lipomyces starkeyi), Cryptococcus sp. (e.g.,Cryptococcus curvatus, aka Apiotricum curvatum), Cunninghamella sp.(e.g., Cunninghamella echinulata), Yarrowia sp. (e.g., Yarrowialipolytica) and yeast (e.g., Saccharomyces sp. (e.g., Saccharomycescerevisiae), Phaffia rhodozyma, Xanthophyllomyces dendrohous, Candidasp. (e.g., Candida utilus); (3) algae, including but not limited to,green algae (e.g., Haematococcus pluvialus, Chlorococcum, Spongiococcum,Neospongiococcum, Dunaliella), Crypthecodinium cohnii, Porphyridiumcruentum, Phaeodactylum tricornicum, Nannochloropsis oculata, Isochrysisgalbana, Chlorella sp.; (4) bacteria, including, but not limited to,blue-green (e.g., Spirulina, Synechococcus, Synechocystis), Escherichiacoli, Flavobacterium, Paracoccus, Erwinia, Agrobacterium, Rhodococcus,Mycobacterium, Streptomyces, Thodococcus, Nocardia, Pseudomonas; and (5)members of the order, Thraustochytriales, including but not limited to:Thraustochytrium sp. (e.g., including former Ulkenia species such as U.visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U.minuta and Ulkenia sp. BP-5601, and including Thraustochytrium striatum,Thraustochytrium aureum, and Thraustochytrium roseum); Labyrinthuloides,Japonochytrium (e.g., Japonochytrium sp.), and Schizochytrium (e.g.,Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium limacinum,Schizochytrium minutum).

According to the present invention, the term “transformed” or“transformation” is used to refer to any method by which an exogenousnucleic acid molecule (i.e., a recombinant nucleic acid molecule) can beinserted into the cell. In microbial systems, the term “transformation”is used to describe an inherited change due to the acquisition ofexogenous nucleic acids by the microorganism and can be essentiallysynonymous with the term “transfection”, which is more commonly used inreference to the similar process in animal cells. The term“transformation” is preferably used herein to refer to the introductionof nucleic acid molecules into microbial cells, such as bacteria andyeast, or into plant cells. Therefore, transformation techniquesinclude, but are not limited to, transfection, electroporation,microinjection, lipofection, biolistic methods (particle bombardment),adsorption, Agrobacterium-mediated transformation, infection andprotoplast fusion. Methods of transforming prokaryotic and eukaryotichost cells are well known in the art. See, e.g., Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982),Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. (1989), incorporated herein by reference in its entirety. Apreferred method for transforming members of the orderThraustochytriales is described in U.S. patent application Ser. No.10/124,807, filed Apr. 16, 2002, incorporated by reference in itsentirety.

Numerous methods for plant transformation have been developed, includingbiological and physical transformation protocols. See, for example, Mikiet al., “Procedures for Introducing Foreign DNA into Plants” in Methodsin Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson,J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition,vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. See, for example, Horsch et al., Science 227:1229 (1985).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references, including Gruber et al., supra, Miki etal., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S.Pat. Nos. 4,940,838 and 5,464,763, each of which is incorporated hereinby reference in its entirety.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles. The expression vector is introduced intoplant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992), each of which isincorporated herein by reference in its entirety.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987), each of which isincorporated herein by reference in its entirety. Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982),each of which is incorporated herein by reference in its entirety.Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994), each of which is incorporated hereinby reference in its entirety.

In one embodiment, an isolated FAS protein of the present invention isproduced by culturing a cell that expresses the protein under conditionseffective to produce the protein, and recovering the protein. Apreferred cell to culture is a recombinant cell of the presentinvention. Effective culture conditions include, but are not limited to,effective media, bioreactor, temperature, pH and oxygen conditions thatpermit protein production. An effective medium refers to any medium inwhich a cell is cultured to produce a FAS protein of the presentinvention. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and Petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant host cell; be secreted into the culture medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cellmembrane. The phrase “recovering the protein” refers to collecting thewhole culture medium containing the protein and need not implyadditional steps of separation or purification. Proteins of the presentinvention can be purified, if desired, using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization. Ifproteins of the present invention are purified, they are preferablyretrieved in “substantially pure” form. As used herein, “substantiallypure” refers to a purity that allows for the effective use of theprotein as a biocatalyst or other reagent.

To produce significantly high yields of short chain fatty acids by themethods of the present invention, a microorganism or plant (or part of aplant, e.g., seeds, pollen, embryos, flowers, fruits, shoots, leaves,roots, stems, explants, etc.) can be genetically modified to increasethe action of the FAS protein of the present invention, and preferably,to enhance production of the FAS protein, and thereby, a short chainfatty acid endproduct.

In a preferred embodiment of the invention, a microorganism thatcontains an endogenous FAS protein of the invention (e.g.,Schizochytrium) is genetically modified to increase or reduce theexpression and activity of the FAS protein. Without being bound bytheory, the present inventors believe that attenuation of the expressionor activity of the endogenous FAS protein in Thraustochytrids such asSchizochytrium will increase the accumulation of the highly desirablepolyunsaturated fatty acids (PUFAs) by the organism, the synthesis ofwhich proceeds using the PUFA polyketide synthase (PKS) system, whichshares some of the same substrates with the FAS system (this PUFA PKSsystem is described in detail in PCT Publication No. WO 02/083870,published Oct. 24, 2002, which is incorporated herein by reference inits entirety). Therefore, decreasing the expression or activity of theFAS in the microorganism will increase the production of PUFAs.

As used herein, a genetically modified microorganism, such as agenetically modified bacterium, protist, microalga, fungus, or othermicrobe, and particularly, any member of the genera of the orderThraustochytriales (e.g., a Thraustochytrid) described herein (e.g.,Schizochytrium, Thraustochytrium, Japonochytrium, Labyrinthuloides), hasa genome which is modified (i.e., mutated or changed) from its normal(i.e., wild-type or naturally occurring) form such that the desiredresult is achieved (i.e., increased or modified FAS expression and/oractivity, production of a desired product using the FAS protein, ordecreased or modified FAS expression and/or activity). Geneticmodification of a microorganism can be accomplished using classicalstrain development and/or molecular genetic techniques. Such techniquesare generally disclosed for microorganisms, for example, in Sambrook etal., 1989, supra, incorporated by reference herein in its entirety. Agenetically modified microorganism can include a microorganism in whichnucleic acid molecules have been inserted, deleted or modified (i.e.,mutated; e.g., by insertion, deletion, substitution, and/or inversion ofnucleotides), in such a manner that such modifications provide thedesired effect within the microorganism.

Preferred microorganism host cells to modify according to the presentinvention include, but are not limited to, any bacteria, protist,microalga, fungus, or protozoa. In one aspect, preferred microorganismsto genetically modify include, but are not limited to, any microorganismof the order Thraustochytriales. Particularly preferred host cells foruse in the present invention could include microorganisms from a genusincluding, but not limited to: Thraustochytrium, Labyrinthuloides,Japonochytrium, and Schizochytrium. Preferred species within thesegenera include, but are not limited to: any Schizochytrium species,including Schizochytrium aggregatum, Schizochytrium limacinum,Schizochytrium minutum; any Thraustochytrium species (including formerUlkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U.profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and includingThraustochytrium striatum, Thraustochytrium aureum, Thraustochytriumroseum; and any Japonochytrium species. Particularly preferred strainsof Thraustochytriales include, but are not limited to: Schizochytriumsp. (S31)(ATCC 20888); Schizochytrium sp. (S8)(ATCC 20889);Schizochytrium sp. (LC-RM)(ATCC 18915); Schizochytrium sp. (SR21);Schizochytrium aggregatum (Goldstein et Belsky)(ATCC 28209);Schizochytrium limacinum (Honda et Yokochi)(IFO 32693); Thraustochytriumsp. (23B)(ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC24473); Thraustochytrium aureum (Goldstein)(ATCC 34304);Thraustochytrium roseum (Goldstein)(ATCC 28210); and Japonochytrium sp.(L1)(ATCC 28207). Other examples of suitable host microorganisms forgenetic modification include, but are not limited to, yeast includingSaccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeastsuch as Candida, Kluyveromyces, or other fungi, for example, filamentousfungi such as Aspergillus, Neurospora, Penicillium, etc. Bacterial cellsalso may be used as hosts. This includes Escherichia coli, which can beuseful in fermentation processes. Alternatively, a host such as aLactobacillus species or Bacillus species can be used as a host.

As used herein, a genetically modified plant can include any geneticallymodified plant including higher plants and particularly, any consumableplants or plants useful for producing a desired product of the presentinvention (e.g., short chain fatty acids or any other lipid product).Such a genetically modified plant has a genome which is modified (i.e.,mutated or changed) from its normal (i.e., wild-type or naturallyoccurring) form such that the desired result is achieved (i.e.,increased or modified FAS expression and/or activity and/or productionof a desired product using the FAS protein). Genetic modification of aplant can be accomplished using classical strain development and/ormolecular genetic techniques. Methods for producing a transgenic plant,wherein a recombinant nucleic acid molecule encoding a desired aminoacid sequence is incorporated into the genome of the plant, are known inthe art and have been described briefly above. A preferred plant togenetically modify according to the present invention is preferably aplant suitable for consumption by animals, including humans.

Preferred plants to genetically modify according to the presentinvention (i.e., plant host cells) include, but are not limited to anyhigher plants, and particularly consumable plants, including crop plantsand especially plants used for their oils. Such plants can include, forexample: canola, soybeans, rapeseed, linseed, corn, safflowers, flax,sunflowers, tobacco, rice, tomatoes and carrots. Other preferred plantsinclude those plants that are known to produce compounds used aspharmaceutical agents, flavoring agents, neutraceutical agents,functional food ingredients or cosmetically active agents or plants thatare genetically engineered to produce these compounds/agents.

According to the present invention, a genetically modified microorganismor plant includes a microorganism or plant that has been modified usingrecombinant technology. As used herein, genetic modifications thatresult in a decrease in gene expression, in the function of the gene, orin the function of the gene product (i.e., the protein encoded by thegene) can be referred to as inactivation (complete or partial),attenuation, deletion, interruption, blockage or down-regulation of agene. For example, a genetic modification in a gene which results in adecrease in the function of the protein encoded by such gene, can be theresult of a complete deletion of the gene (i.e., the gene does notexist, and therefore the protein does not exist), a mutation in the genewhich results in incomplete or no translation of the protein (e.g., theprotein is not expressed), or a mutation in the gene or introduction ofa regulatory sequence or protein into the host which decreases,attenuates or abolishes the expression and/or the natural function ofthe protein (e.g., a protein is expressed which has decreased or noenzymatic activity or action). In the present invention, geneticmodifications that decrease the expression or activity of a FAS arepreferably not complete inactivations, as such mutations are lethal inmicroorganisms. Preferably, such modifications reduce or attenuate, butdo not entirely delete, the expression or function of a FAS protein.Genetic modifications that result in an increase in gene expression orfunction can be referred to as amplification, overproduction,overexpression, activation, enhancement, addition, or up-regulation of agene.

In one embodiment of the present invention, a genetic modification of amicroorganism or plant increases or decreases the expression and/oractivity of a FAS protein of the present invention. Such a geneticmodification includes any type of modification and specifically includesmodifications made by recombinant technology and/or by classicalmutagenesis. It should be noted that reference to increasing the action(activity) of FAS refers to any genetic modification in themicroorganism or plant in question and/or in the recombinant nucleicacids containing the FAS-encoding DNA with which the organism istransformed that results in increased functionality of the protein andcan include higher activity of the protein (e.g., specific activity orin vivo enzymatic activity), reduced inhibition or degradation of theprotein, and overexpression of the protein. For example, gene copynumber can be increased, expression levels can be increased by use of apromoter that gives higher levels of expression than that of the nativepromoter, or a gene can be altered by genetic engineering or classicalmutagenesis to increase the action of an enzyme. In one aspect, FASactivity or expression can be modified by modifying a nucleic acid orprotein that interacts with a FAS gene or protein and normally modulatesthe expression or activity of the FAS gene or protein. Such amodification can be achieved by recombinant or classical mutationaltechniques.

Similarly, reference to decreasing the action (activity) of a FASprotein refers to any genetic modification in the microorganism or plantin question and/or in the recombinant nucleic acids containing theFAS-encoding DNA (including FAS regulatory regions or inhibitorsthereof) with which the organism is transformed that results indecreased functionality of the enzymes and includes decreased activityof the enzymes (e.g., specific activity), increased inhibition ordegradation of the enzymes and a reduction or elimination of expressionof the enzyme. For example, the action of FAS of the present inventioncan be decreased by blocking or reducing the production of the protein,“knocking out” all or a portion of the gene encoding the protein,reducing FAS activity, or inhibiting the activity of FAS (any one, twoor more of the biological activities of a FAS of the invention).Blocking or reducing the production of an enzyme can include placing thegene encoding the protein under the control of a heterologous promoterthat requires the presence of an inducing compound in the growth medium.By establishing conditions such that the inducer becomes depleted fromthe medium, the expression of the gene encoding the protein (andtherefore, of protein synthesis) could be turned off or on as desired.Blocking or reducing the activity of a protein could also include usingan excision technology approach similar to that described in U.S. Pat.No. 4,743,546, incorporated herein by reference in its entirety. To usethis approach, the gene encoding the protein of interest is clonedbetween specific genetic sequences that allow specific, controlledexcision of the gene from the genome. Excision could be prompted by, forexample, a shift in the cultivation temperature of the culture, as inU.S. Pat. No. 4,743,546, or by some other physical or nutritionalsignal. PCT Publication No. WO 02/083869, published Oct. 24, 2002,describes methods for inactivation of genes in Thraustochytriales, andis incorporated herein by reference in its entirety.

In one embodiment of the present invention, it is contemplated that amutagenesis program could be combined with a selective screening processto obtain microorganisms of interest. The mutagenesis methods couldinclude, but are not limited to: chemical mutagenesis, gene shuffling,switching regions of the genes encoding specific enzymatic domains, ormutagenesis restricted to specific regions of those genes, as well asother methods. For example, high throughput mutagenesis methods could beused to influence or optimize production of the desired fatty acids orother lipid products. Such methods could be combined with selective(i.e., targeted or directed) modification of the FAS by molecularbiology techniques. For example, one could use selective modificationtechniques to modify a microorganism, for example, by introduction of arecombinant nucleic acid molecule encoding the FAS protein of theinvention into any suitable host cell, including host cells comprisingan endogenous FAS, and then use mutagenesis technologies to optimizefatty acid production and to create strains having improved fatty acidsynthesis activity or to select for microorganisms with other improvedor desired qualities. Screening methods are also useful for identifyingother organisms having homologous FAS genes to the FAS ofSchizochytrium. Homologous FAS genes identified in such organisms can beused in methods similar to those described herein.

In one embodiment of the present invention, a genetically modifiedmicroorganism or plant includes a microorganism or plant that has anenhanced ability to synthesize fatty acids in general or an enhancedability to synthesize specific short chain fatty acids. According to thepresent invention, “an enhanced ability to synthesize” a product refersto any enhancement, or up-regulation, in a pathway related to thesynthesis of the product such that the microorganism or plant producesan increased amount of the product compared to the wild-typemicroorganism or plant, cultured or grown, under the same conditions. Inone embodiment of the present invention, enhancement of the ability of amicroorganism or plant to synthesize short chain fatty acids isaccomplished by amplification of the expression of the FAS gene.Amplification of the expression of FAS can be accomplished in anysuitable host cell (e.g., a Thraustochytriales cell, a bacterial cell, ayeast cell, a plant cell), for example, by introduction of a recombinantnucleic acid molecule encoding the FAS gene, or by modifying regulatorycontrol over a native FAS gene, in the case of Thraustochytriales.

According to the present invention, “selective modification” of anorganism or nucleic acid molecule refers to a targeted, or directed,modification, where the modification to be made is predetermined anddesigned, for example, by knowledge of the gene structure of the FAS ofthe present invention. For example, selective modification of anorganism can be achieved by introduction (e.g., overexpression) of arecombinant nucleic acid molecule encoding a FAS protein, or by targetedmodification of an endogenous gene, such as by homologous recombination.Selective modification is distinguished from random mutagenesistechniques, where in the latter process, the mutation is randomlygenerated by a non-target-specific method and the desired phenotype issubsequently selected through screening of mutants for the phenotype.Selective modification techniques and classical random mutagenesis andscreening techniques can be combined in the present invention to producea variety of genetically modified organisms.

Therefore, it is an embodiment of the present invention to provide amicroorganism or plant that is transformed with a recombinant nucleicacid molecule comprising a nucleic acid sequence encoding FAS of thepresent invention. Preferred recombinant nucleic acid moleculescomprising such a nucleic acid sequence include recombinant nucleic acidmolecules comprising any of the FAS nucleic acid sequences previouslydescribed herein. It is one embodiment of the present invention toprovide a microorganism or plant which is transformed with a geneticallymodified recombinant nucleic acid molecule comprising a nucleic acidsequence encoding a mutant, or homologue, FAS. Protein homologues havebeen described in detail herein.

It is another embodiment of the present invention to provide agenetically modified microorganism for producing a fatty acid by abiosynthetic process, wherein the microorganism comprises a nucleic acidmolecule encoding a FAS protein of the present invention and wherein thenucleic acid molecule encoding the FAS protein has been modified toincrease the expression or biological activity of the FAS. The FAS canbe any FAS described herein, including homologues and biologicallyactive fragments as described herein. In one aspect of the invention,the microorganism has an endogenous FAS (e.g., a member ofThraustochytriales), and the endogenous gene is modified to increase theexpression or activity of the FAS (e.g., by introducing a promoter thatgives higher levels of expression than that of the native promoter, bygenetically mutating the endogenous gene to increase the activity of theenzyme, etc.). In another embodiment, the microorganism is geneticallymodified by transformation with a recombinant nucleic acid moleculeencoding a FAS of the invention. Such a microorganism can be anysuitable host microorganism and in one embodiment, is aThraustochytriales microorganism (e.g., a Schizochytrium), such that themicroorganism comprises both an endogenous FAS and a recombinant FAS.The FAS proteins in this scenario need not be identical, since one orboth of the endogenous and recombinant FAS proteins can be modified ascompared to a wild-type Schizochytrium FAS disclosed herein to produce aFAS homologue. For example, one or both of the endogenous or recombinantFAS-encoding nucleic acid molecules can be modified to increase theexpression or activity of the FAS proteins.

Accordingly, one embodiment of the invention is a biomass comprising anyof the microorganisms described herein comprising a nucleic acidmolecule encoding a FAS of the present invention that has been modifiedto increase the expression or biological activity of the FAS asdescribed above. As used herein, a biomass refers to a population ofmicrobial cells that have been harvested from a fermentation or cultureprocess. Various fermentation parameters for inoculating, growing andrecovering microfloral biomasses are discussed in detail in U.S. Pat.No. 5,130,242, incorporated herein by reference in its entirety. Thebiomass harvested from a fermentation run can be dried (e.g., spraydrying, tunnel drying, vacuum drying, or a similar process) and used inany food, pharmaceutical or other desired product. Alternatively, theharvested and washed biomass can be used directly (without drying) invarious products. To extend its shelf life, the wet biomass can beacidified (approximate pH=3.5-4.5) and/or pasteurized or flash heated toinactivate enzymes and then canned, bottled or packaged under a vacuumor non-oxidizing atmosphere (e.g., N₂ or CO₂).

One embodiment of the present invention is a method to produce a shortchain fatty acid by a biosynthetic process, comprising culturing in afermentation medium a genetically modified microorganism that hasincreased expression or biological activity of a FAS protein asdescribed above. For example, the microorganism can have increasedexpression or biological activity of any FAS proteins described herein,including homologues and enzymatically active portions thereof. The FASprotein can be an endogenous FAS protein and/or a recombinant FASprotein according to the invention. The microorganism is cultured orgrown in a suitable medium, under conditions effective to produce thedesired fatty acid or other lipid product. An appropriate, or effective,medium refers to any medium in which a genetically modifiedmicroorganism of the present invention, when cultured, is capable ofproducing the desired product. Such a medium is typically an aqueousmedium comprising assimilable carbon, nitrogen and phosphate sources.Such a medium can also include appropriate salts, minerals, metals andother nutrients. Microorganisms of the present invention can be culturedin conventional fermentation bioreactors. The microorganisms can becultured by any fermentation process which includes, but is not limitedto, batch, fed-batch, cell recycle, and continuous fermentation.Preferred growth conditions for potential host microorganisms accordingto the present invention are well known in the art. The desired productsproduced by the genetically modified microorganism can be recovered fromthe fermentation medium using conventional separation and purificationtechniques. For example, the fermentation medium can be filtered orcentrifuged to remove microorganisms, cell debris and other particulatematter, and the product can be recovered from the cell-free supernatantby conventional methods, such as, for example, ion exchange,chromatography, extraction, solvent extraction, membrane separation,electrodialysis, reverse osmosis, distillation, chemical derivatizationand crystallization. Alternatively, microorganisms producing the desiredproduct, or extracts and various fractions thereof, can be used withoutremoval of the microorganism components from the product, such as in abiomass of the invention.

One embodiment of the present invention is a method to produce shortchain fatty acids by growing or culturing a genetically modified plantof the present invention as previously described herein. Such a methodincludes the step of culturing in a fermentation medium or growing in asuitable environment, such as soil, a plant having a geneticmodification to increase the action of FAS. Preferably, the geneticmodification includes transformation or transfection of the plant with arecombinant nucleic acid molecule that expresses a protein having FASbiological activity. Such a protein can include any of the FAS proteinsdescribed herein, including any homologue of a naturally occurring FAShaving biological activity.

In the method for production of short chain fatty acids of the presentinvention, a plant that has a genetic modification to increase theaction of FAS is cultured in a fermentation medium or grown in asuitable medium such as soil for production of the FAS. An appropriate,or effective, fermentation medium has been discussed in detail above. Asuitable growth medium for higher plants includes any growth medium forplants, including, but not limited to, soil, sand, any other particulatemedia that support root growth (e.g. vermiculite, perlite, etc.) orHydroponic culture, as well as suitable light, water and nutritionalsupplements which optimize the growth of the higher plant. Thegenetically modified plants of the present invention are engineered toproduce significant quantities of short chain fatty acids throughincreased action of the FAS protein of the present invention. The shortchain fatty acids can be recovered through purification processes whichextract these products from the plant. In a preferred embodiment, thefatty acids are recovered by harvesting the plant or plant fraction(e.g., oils). In this embodiment, the plant or plant fraction can beconsumed in its natural state or further processed into consumableproducts.

Another embodiment of the invention relates to a genetically modifiedmicroorganism with a reduced ability to produce short chain fatty acids(resulting in reduced amounts of short chain fatty acid production inthe microorganism), wherein the microorganism has been geneticallymodified to selectively delete, but more preferably attenuate (inhibit,reduce the expression or activity of, etc., without actually deleting orcompletely inactivating the gene or its protein product) a FAS gene orportion thereof encoding a functional domain. In a preferred embodiment,the microorganism is a microalga, and in a more preferred embodiment, isa Thraustochytriales microorganism (e.g., a Schizochytrium). The FASgene can be modified by modification to the coding region of the FASgene or to a regulatory region of the FAS gene, such that expressionand/or biological activity of the FAS gene is reduced, and preferablyinhibited so that the microorganism has reduced production of shortchain fatty acids and more preferably, increased production of longchain fatty acids, and particularly, of polyunsaturated fatty acids(PUFAs). In one embodiment the FAS gene is partially or completelydeleted or inactivated, including by replacing the gene with a non-FASnucleic acid sequence, such as by gene disruption through homologousrecombination. In this aspect, the FAS gene is mutated or inactivated(or deleted) by targeted homologous recombination with a nucleic acidsequence that hybridizes to the FAS gene that includes a heterologousnucleic acid sequence that disrupts the coding region of the FAS gene.

Production of a microorganism that has reduced FAS expression oractivity has commercial benefits, as described above. Microorganismsthat contain the FAS of the present invention include members ofThraustochytriales, which are known to be valuable organisms for theproduction of lipids containing high levels of polyunsaturated fattyacids (PUFAs), including highly unsaturated fatty acids such as omega-3fatty acids. Polyunsaturated fatty acids (PUFAs) are critical componentsof membrane lipids in most eukaryotes (Lauritzen et al., Prog. LipidRes. 40 1 (2001); McConn et al., Plant J. 15, 521 (1998)) and areprecursors of certain hormones and signaling molecules (Heller et al.,Drugs 55, 487 (1998); Creelman et al., Annu. Rev. Plant Physiol. PlantMol. Biol. 48, 355 (1997)). According to the present invention, apreferred PUFA is a long chain PUFA, which is defined as a PUFA havingeighteen carbons or more. PUFAs include any omega-3 or omega-6polyunsaturated fatty acids with three or more double bonds. Omega-3PUFAs are polyethylenic fatty acids in which the ultimate ethylenic bondis three carbons from and including the terminal methyl group of thefatty acid and include, for example, docosahexaenoic acid C22:6(n-3)(DHA) and omega-3 docosapentaenoic acid C22:5(n-3) (DPAn-3). Omega-6PUFAs are polyethylenic fatty acids in which the ultimate ethylenic bondis six carbons from and including the terminal methyl group of the fattyacid and include, for example, arachidonic acid C20:4(n-6) (ARA),C22:4(n-6), omega-6 docosapentaenoic acid C22:5(n-6) (DPAn-6) anddihomogammalinolenic acid C20:3(n-6)(dihomo GLA). Members ofThraustochytriales, such as Schizochytrium, accumulate large quantitiesof triacylglycerols rich in PUFAs. Since these lipid products are usefulin a variety of food and other commercial products, it would be usefulto enhance the ability of microorganisms to preferentially produce thePUFAs. The present invention provides one method by which this goal canbe achieved (i.e., by attenuation of the competing FAS system).

Accordingly, another embodiment of the invention relates to a biomasscomprising genetically modified microorganism (e.g., a microorganism ofthe order Thraustochytriales (e.g., Schizochytrium, Thraustochytrium))that have reduced short chain fatty acid synthesis and more preferably,increased PUFA synthesis, as compared to a wild-type microorganism ofthe same species, as described above. It is to be understood thatorganisms other than Thraustochytriales may be discovered which containa FAS protein having homology and most or all of the biologicalactivities of the full length FAS described herein. Such microorganismscan also be modified to reduce the expression or activity of the FASsystem, particularly if such microorganisms are also useful forproducing PUFAs.

Fatty acids produced in accordance with the methods of the presentinvention are typically produced as lipids. As used herein, the term“lipid” includes phospholipids (PL); free fatty acids; esters of fattyacids; triacylglycerols (TAG); diacylglycerides; phosphatides; sterolsand sterol esters; carotenoids; xanthophylls (e.g., oxycarotenoids);hydrocarbons; and other lipids known to one of ordinary skill in theart. The term “fatty acid” (including as the term is used in“polyunsaturated fatty acid” and “PUFA”) includes not only the freefatty acid form, but other forms as well, such as the triacyglycerol(TAG) form and the phospholipids (PL) form.

A food product, as used herein, includes any food ingredient (e.g., afood product that is part of another food product, such as an oil), andalso includes, but is not limited to: fine bakery wares, bread androlls, breakfast cereals, processed and unprocessed cheese, condiments(ketchup, mayonnaise, etc.), dairy products (milk, yoghurt), puddingsand gelatine desserts, carbonated drinks, teas, powdered beverage mixes,processed fish products, fruit-based drinks, chewing gum, hardconfectionery, frozen dairy products, processed meat products, nut andnut-based spreads, pasta, processed poultry products, gravies andsauces, potato chips and other chips or crisps, chocolate and otherconfectionery, soups and soup mixes, soya based products (milks, drinks,creams, whiteners), vegetable oil-based spreads, and vegetable-baseddrinks. Other products include dietary supplements, a pharmaceuticalformulation (e.g., a pharmaceutical product), humanized animal milk, andinfant formulas. Suitable pharmaceutical formulations or productsinclude, but are not limited to, an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, a cholesterol loweringformulation, and products used to treat a condition selected from thegroup consisting of: chronic inflammation, acute inflammation,gastrointestinal disorder, cancer, cachexia, cardiac restenosis,neurodegenerative disorder, degenerative disorder of the liver, bloodlipid disorder, osteoporosis, osteoarthritis, autoimmune disease,preeclampsia, preterm birth, age related maculopathy, pulmonarydisorder, and peroxisomal disorder.

Therefore, another embodiment of the present invention relates to amethod for producing lipids, and preferably PUFAs, from a biosyntheticprocess, comprising culturing under conditions effective to produce thelipids genetically modified microorganisms (e.g., of the orderThraustochytriales) as previously described herein, wherein themicroorganisms have been genetically modified to selectively increase ordecrease (depending on the goal) a FAS gene as described above. Thelipids can be recovered using any one of a variety of recoverytechniques known in the art or the entire microorganism or extractsthereof can be recovered. One aspect of the invention relates to amethod for recovering lipids from a biosynthetic process, comprisingrecovering lipids from a culture of genetically modified microorganisms(e.g., of the order Thraustochytriales), wherein the microorganisms havebeen genetically modified to selectively increase or decrease a FAS geneas described above. Techniques for recovery of lipids from the cultureare known in the art and include, but are not limited to, ion exchange,chromatography, extraction, solvent extraction, phase separation,membrane separation, electrodialysis, reverse osmosis, distillation,chemical derivatization and crystallization.

Another embodiment of the present invention is a method for producingshort chain fatty acids using an isolated FAS, including a homologue ofa FAS as described herein. The method can be operated in batch orcontinuous mode using a stirred tank, a plug-flow column reactor orother apparatus known to those skilled in the art.

In one embodiment, the FAS is bound to a solid support, i.e., animmobilized enzyme. As used herein, a FAS bound to a solid support(i.e., an immobilized FAS) includes immobilized isolated FAS,immobilized cells which contain a FAS (including immobilized andgenetically modified Thraustochytriales, bacterial, fungal (e.g.,yeast), microalgal, or plant cells), stabilized intact cells andstabilized cell/membrane homogenates. Stabilized intact cells andstabilized cell/membrane homogenates include cells and homogenates fromnaturally occurring microorganisms expressing FAS or from geneticallymodified microorganisms or plants as disclosed elsewhere herein. Thus,although methods for immobilizing FAS are discussed below, it will beappreciated that such methods are equally applicable to immobilizingcells and in such an embodiment, the cells can be lysed.

A variety of methods for immobilizing an enzyme are disclosed inIndustrial Enzymology 2nd Ed., Godfrey, T. and West, S. Eds., StocktonPress, New York, N.Y., 1996, pp. 267-272; Immobilized Enzymes, Chibata,I. Ed., Halsted Press, New York, N.Y., 1978; Enzymes and ImmobilizedCells in Biotechnology, Laskin, A. Ed., Benjamin/Cummings PublishingCo., Inc., Menlo Park, Calif., 1985; and Applied Biochemistry andBioengineering, Vol. 4, Chibata, I. and Wingard, Jr., L. Eds, AcademicPress, New York, N.Y., 1983, which are incorporated herein in theirentirety.

Briefly, a solid support refers to any solid organic supports,artificial membranes, biopolymer supports, or inorganic supports thatcan form a bond with FAS (or cell) without significantly affecting theactivity of isolated FAS. Exemplary organic solid supports includepolymers such as polystyrene, nylon, phenol-formaldehyde resins, acryliccopolymers (e.g., polyacrylamide), stabilized intact whole cells, andstabilized crude whole cell/membrane homogenates. Exemplary biopolymersupports include cellulose, polydextrans (e.g., Sephadex®), agarose,collagen and chitin. Exemplary inorganic supports include glass beads(porous and nonporous), stainless steel, metal oxides (e.g., porousceramics such as ZrO₂, TiO₂, Al₂O₃, and NiO) and sand. Preferably, thesolid support is selected from the group consisting of stabilized intactcells and/or crude cell homogenates. Preparation of such supportsrequires a minimum of handling and cost. Additionally, such supportsprovide excellent stability of the enzyme.

Stabilized intact cells and/or cell/membrane homogenates can beproduced, for example, by using bifunctional crosslinkers (e.g.,glutaraldehyde) to stabilize cells and cell homogenates. In both theintact cells and the cell membranes, the cell wall and membranes act asimmobilizing supports. In such a system, integral membrane proteins arein the “best” lipid membrane environment. Whether starting with intactcells or homogenates, in this system the cells are either no longer“alive” or “metabolizing”, or alternatively, are “resting” (i.e., thecells maintain metabolic potential and active FAS, but under the cultureconditions are not growing); in either case, the immobilized cells ormembranes serve as biocatalysts.

FAS can be bound to a solid support by a variety of methods includingadsorption, cross-linking (including covalent bonding), and entrapment.Adsorption can be through van der Waal's forces, hydrogen bonding, ionicbonding, or hydrophobic binding. Exemplary solid supports for adsorptionimmobilization include polymeric adsorbents and ion-exchange resins.Solid supports in a bead form are particularly well-suited. The particlesize of an adsorption solid support can be selected such that theimmobilized enzyme is retained in the reactor by a mesh filter while thesubstrate (e.g., the precursor or substrate used as a starting materialto produce the desired fatty acid) is allowed to flow through thereactor at a desired rate. With porous particulate supports it ispossible to control the adsorption process to allow FAS or geneticallymodified microorganism cells to be embedded within the cavity of theparticle, thus providing protection without an unacceptable loss ofactivity.

Cross-linking of a FAS to a solid support involves forming a chemicalbond between a solid support and a FAS. It will be appreciated thatalthough cross-linking generally involves linking a FAS to a solidsupport using an intermediary compound, it is also possible to achieve acovalent bonding between the enzyme and the solid support directlywithout the use of an intermediary compound. Cross-linking commonly usesa bifunctional or multifunctional reagent to activate and attach acarboxyl group, amino group, sulfur group, hydroxy group or otherfunctional group of the enzyme to the solid support. The term “activate”refers to a chemical transformation of a functional group which allows aformation of a bond at the functional group. Exemplary amino groupactivating reagents include water-soluble carbodiimides, glutaraldehyde,cyanogen bromide, N-hydroxysuccinimide esters, triazines, cyanuricchloride, and carbonyl diimidazole. Exemplary carboxyl group activatingreagents include water-soluble carbodiimides, andN-ethyl-5-phenylisoxazolium-3-sulfonate. Exemplary tyrosyl groupactivating reagents include diazonium compounds. And exemplarysulfhydryl group activating reagents includedithiobis-5,5′-(2-nitrobenzoic acid), and glutathione-2-pyridyldisulfide. Systems for covalently linking an enzyme directly to a solidsupport include Eupergit®, a polymethacrylate bead support availablefrom Rohm Pharma (Darmstadt, Germany), kieselguhl (Macrosorbs),available from Sterling Organics, kaolinite available from English ChinaClay as “Biofix” supports, silica gels which can be activated bysilanization, available from W. R. Grace, and high-density alumina,available from UOP (Des Plaines, Ill.).

Entrapment can also be used to immobilize FAS. Entrapment of FASinvolves formation of, inter alia, gels (using organic or biologicalpolymers), vesicles (including microencapsulation), semipermeablemembranes or other matrices. Exemplary materials used for entrapment ofan enzyme include collagen, gelatin, agar, cellulose triacetate,alginate, polyacrylamide, polystyrene, polyurethane, epoxy resins,carrageenan, and egg albumin. Some of the polymers, in particularcellulose triacetate, can be used to entrap the enzyme as they are spuninto a fiber. Other materials such as polyacrylamide gels can bepolymerized in solution to entrap the enzyme. Still other materials suchas polyglycol oligomers that are functionalized with polymerizable vinylend groups can entrap enzymes by forming a cross-linked polymer with UVlight illumination in the presence of a photosensitizer.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies, or antigen binding fragments thereof, thatare capable of selectively binding to a FAS protein of the presentinvention (e.g., FAS antibodies). The phrase “selectively binds” refersto the specific binding of one protein to another (e.g., an antibody,fragment thereof, or binding partner to an antigen), wherein the levelof binding, as measured by any standard assay (e.g., an immunoassay), isstatistically significantly higher than the background control for theassay. For example, when performing an immunoassay, controls typicallyinclude a reaction well/tube that contain antibody or antigen bindingfragment alone (i.e., in the absence of antigen), wherein an amount ofreactivity (e.g., non-specific binding to the well) by the antibody orantigen binding fragment thereof in the absence of the antigen isconsidered to be background. Binding can be measured using a variety ofmethods standard in the art including enzyme immunoassays (e.g., ELISA),immunoblot assays, etc.). Antibodies of the present invention can bepolyclonal or monoclonal, functional equivalents such as antibodyfragments and genetically-engineered antibodies, including single chainantibodies or chimeric antibodies, including bi-specific antibodies thatcan bind to more than one epitope.

Generally, in the production of an antibody, a suitable experimentalanimal, such as, for example, but not limited to, a rabbit, a sheep, ahamster, a guinea pig, a mouse, a rat, or a chicken, is exposed to anantigen against which an antibody is desired. Typically, an animal isimmunized with an effective amount of antigen that is injected into theanimal. An effective amount of antigen refers to an amount needed toinduce antibody production by the animal. The animal's immune system isthen allowed to respond over a pre-determined period of time. Theimmunization process can be repeated until the immune system is found tobe producing antibodies to the antigen. In order to obtain polyclonalantibodies specific for the antigen, serum is collected from the animalthat contains the desired antibodies (or in the case of a chicken,antibody can be collected from the eggs). Such serum is useful as areagent. Polyclonal antibodies can be further purified from the serum(or eggs) by, for example, treating the serum with ammonium sulfate.

Monoclonal antibodies may be produced according to the methodology ofKohler and Milstein (Nature 256:495-497, 1975). For example, Blymphocytes are recovered from the spleen (or any suitable tissue) of animmunized animal and then fused with myeloma cells to obtain apopulation of hybridoma cells capable of continual growth in suitableculture medium. Hybridomas producing the desired antibody are selectedby testing the ability of the antibody produced by the hybridoma to bindto the desired antigen.

Genetically engineered antibodies of the invention include thoseproduced by standard recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Particular examples include, chimeric antibodies,where the V_(H) and/or V_(L) domains of the antibody come from adifferent source to the remainder of the antibody, and CDR graftedantibodies (and antigen binding fragments thereof), in which at leastone CDR sequence and optionally at least one variable region frameworkamino acid is (are) derived from one source and the remaining portionsof the variable and the constant regions (as appropriate) are derivedfrom a different source. Construction of chimeric and CDR-graftedantibodies are described, for example, in European Patent Applications:EP-A 0194276, EP-A 0239400, EP-A 0451216 and EP-A 0460617.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example describes the cloning and characterization of theSchizochytrium FAS proteins of the invention.

Briefly, a cDNA library was prepared from mRNA isolated fromSchizochytrium cells as described in Metz et al., Science 293, pp290-292 (2001). Approximately 8,500 clones were randomly chosen andsequenced from the 5′ end using a vector-derived primer. A search ofthis database using various FAS proteins as queries revealed thepresence of many homologues. When the database was queried using theyeast FAS subunits (a and b), over 40 sequences were found withsignificant homology. Most of these Schizochytrium sequences could beassembled into a single large (˜4000 bp) contig. When individual cDNAsequences were used as queries in a BLAST search (tblastn) the bestmatches were, in most cases, to the FAS subunits of fungal organisms. Inthese organisms the Type I FAS is composed of two subunits (a and b),each carrying a distinct set of enzymatic domains. One anomaly of thecDNA sequence data was that all but one of the matches were to the “a”subunit. Using the sequence tags as a guide, the inventors cloned thecorresponding regions of genomic DNA encoding those cDNA-derivedsequences. As the project proceeded, the reason for the anomalousrepresentation of the subunits in the cDNA library became clear. The FASin Schizochytrium is encoded by a single large gene. The FAS Orfcontains 12,408 bp and encodes a protein with a deduced molecular massof 444,884 Daltons. No evidence for introns could be found, either byanalysis of the genomic sequence itself or by comparison to availablecDNA sequences. Blast and Pfam results plus motif analyses were used toestablish a preliminary structure and functional identification of thedomains of this Type I protein. Both in terms of amino acid sequence andin the sequential organization, the Schizochytrium FAS resembles afusion of the head (N-terminus) of the fungal FAS a subunit to the tail(C-terminus) of the b subunit.

The DNA fragments containing the Schizochytrium FAS open reading framewere cloned from a lambda library of genomic DNA. Standard methods wereused to produce that library as well as PCR derived probes (based onsequences obtained from cDNA clones) for isolation of the respective DNAfragments. The Orf encoding the FAS gene was sequenced using acombination of subcloning and primer walking.

The complete Schizochytrium FAS-encoding sequence is a 12,408 nucleotidesequence (not including the stop codon), represented herein by SEQ IDNO:1, which encodes a 4136 amino acid sequence, represented herein asSEQ ID NO:2. Within the Schizochytrium FAS protein are nine domains asdescribed above: (a) one acetyl-transferase (AT) domain (SEQ ID NO:5);(b) one enoyl ACP reductase (ER) domain (SEQ ID NO:6); (c) one dehydrase(DH) domain (SEQ ID NO:7); (d) one malonyl/palmitoyl acyltransferase(M/PAT) domain (SEQ ID NO:8); (e) two acyl carrier protein (ACP) domains(SEQ ID NO:9 and (SEQ ID NO:10); (f) one keto-acyl ACP reductase (KR)domain (SEQ ID NO:11); (g) one keto-acyl ACP synthase (KS) domain (SEQID NO:12); and (h) one phosphopantetheinyl transferase (PPT) domain (SEQID NO:13).

FIG. 1 is a schematic representation of the putative enzymatic domainspresent in the Schizochytrium FAS protein based on the identification,cloning and sequencing of this protein by the present inventors,followed by analysis of the sequences. The size and positions of theputative domains relative to the entire protein, and the identificationof the functions of those domains, are based on Pfam analyses resultsand on homology to the well-characterized FAS (α and β subunits) ofbaker's yeast (Saccharomyces cerevisiae) (references 1, 2 & 3). Thedomain organization and sizes relative to the overall protein sizes ofthe yeast FAS (α and β subunits) are shown for comparison.

Abbreviations: (AT) acetyl-transferase; (ER) enoyl ACP reductase; (DH)dehydrase (DH); (M/PAT) malonyl/palmitoyl acyltransferase; (ACP #1) thefirst acyl carrier protein; (ACP #2) the second acyl carrier protein;(R) keto-acyl ACP reductase; (KS) keto-acyl ACP synthase; and (PPT)phosphopantetheinyl transferase.

REFERENCES

1. Mohamed et al., J Biol Chem. 1988, Sep. 5; 263(25):12315-25.

2. Chirala et al., J Biol Chem. 1987, Mar. 25; 262(9):4231-40.

3. Fichtlscherer et al., Eur J Biochem. 2000, May; 267(9):2666-71.

Example 2

The following example describes the targeted inactivation of theSchizochytrium FAS gene.

The Schizochytrium FAS gene was inactivated by targeted inactivation,using technology that is described in PCT Publication No. WO 02/083869,published Oct. 24, 2002. PCT Publication No. WO 02/083869 isincorporated herein by reference in its entirety.

FIG. 2A diagrams the construction of a plasmid used for targetedinactivation of the Schizochytrium FAS gene. The straight linerepresents the region of the Schizochytrium chromosome containing theFAS gene. The arrow above the line indicates the position of the openreading frame (Orf) that encodes the FAS protein. The boxed area on theline (both striped and solid) represents the portion of the chromosomecloned into the plasmid; JK870. Digestion of JK870 with the restrictionenzyme BglII, followed by dilution and ligation, resulted in recovery ofplasmid JK876, in which the BglII fragment (solid box) has been deleted.A fragment of DNA containing the ble gene (encoding a protein thatconfers resistance to Zeocin™) along with the α-tubulin promoter regionand a SV40 terminator region (see below), isolated from the plasmidpTUBZEO11-2, by digestion with BamHI, is then cloned into the BglII siteof JK876 to generate the plasmid JK874.

FIG. 2B diagrams the events that are believed to occur and result in thestable inactivation of the FAS gene in Schizochytrium. The plasmid JK874is introduced into whole cells of Schizochytrium (either the 20888strain, or the cell wall deficient strain—AC66) via particle bombardment(as described in PCT Publication No. WO 02/083869, ibid.). The initialselection for transformed cells (i.e., having the Zeocin™ resistancegene from plasmid JK874 introduced into the chromosome in a functionalmanner) was made on agar plates with growth medium containing Zeocin™and supplemented with free saturated fatty acids (i.e., C16:0)solubilized with β-cyclodextrin. Colonies that grow under theseselective conditions were transferred to a new plate in a grid-likearray. The transformants were then tested for their ability to growwithout fatty acid supplementation by transferring cells from thecolonies to a plate containing Zeocin™ but lacking the fatty acid. Thecolonies that are Zeocin™ resistant and require supplementation with16:0 fatty acids for growth were presumed to have had the FAS geneinactivated via a homologous recombination event. Those colonies thatdid not show reversion to 16:0 prototrophy were presumed to haveintegrated the Zeocin™ resistance gene cassette into the FAS region viaa double crossover event as diagramed. The stability of the phenotype(requirement for supplementation with 16:0 fatty acid for growth)indicates a portion of the FAS Orf has been replaced with the DNAconferring Zeocin™ resistance. The integration into the FAS portion ofthe chromosome was then confirmed via a PCR reaction as diagramed. Inthis manner, FAS knock-outs in both Schizochytrium strains ATCC 20888and AC66 were obtained.

Example 3

The following example describes in vitro fatty acid synthesis assays instrains in which the Schizochytrium FAS system of the invention has beeninactivated.

FIG. 3 shows the synthesis of fatty acids from [1-¹⁴C]-malonyl-CoA incell free homogenates from a cell wall deficient strain ofSchizochytrium (AC66) and mutants of that strain in which either thePUFA polyketide synthase orfC gene (PUFA-KO) or the FAS gene (FAS-KO)(see FIGS. 2A and 2B above) have been inactivated. Cells were disruptedby sonication in 100 mM phosphate buffer, pH 7.2 containing 2 mM DTT, 1mM EDTA and 10% glycerol. Aliquots of the homogenates were supplementedwith 10 μM acetyl-CoA, 100 μM [1-¹⁴C]-malonyl-CoA, 2 mM NADH and 2 mMNADPH and incubated for 30 min at 30° C. Fatty acids in the reactionmixture were converted to methyl esters, extracted into hexane andseparated by AgNO₃ thin layer chromatography (TLC) (solvent:hexane/diethyl ether/HOAc 70/30/2 by vol). Radioactivity on the TLCplates was detected using a Molecular Dynamics phosphorimaging system.Migration distances of fatty acid methyl ester standards (16:0, 18:1,DPA n-6, and DHA) are indicated to the right side of the diagram.

Assays of the homogenate of the AC66 parent strain reveal the presenceof radiolabeled bands that co-migrate with the 16:0-, 18:1- andDHA-methyl ester standards. In the PUFA-KO mutant strain, theradiolabeled band that co-migrates with the DHA methyl ester standard isgreatly reduced (or lacking). In the FAS-KO strain, radiolabeled bandsthat co-migrate with the 16:0- and 18:1-methyl ester standards (as wellas another band) are greatly reduced (or lacking). These data indicatethat inactivation of the FAS gene results in the loss of the ability tosynthesize short chain saturated fatty acids and suggest that 18:1 isderived from products of the FAS. The data also show that synthesis ofDHA and DPA in Schizochytrium is not dependent on products of the FASsystem.

Each publication cited herein is incorporated herein by reference in itsentirety.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A genetically modified Thraustochytriales microorganism for producingshort chain fatty acids by a biosynthetic process, wherein themicroorganism comprises a nucleic acid molecule encoding a fatty acidsynthase and wherein the nucleic acid molecule encoding the fatty acidsynthase has been modified to increase the expression of the fatty acidsynthase, the fatty acid synthase comprising an amino acid sequenceselected from the group consisting of: a) an amino acid sequenceselected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting ofpositions 1-500 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting ofpositions 3900-4136 of SEQ ID NO:2, and a biologically active fragmentthereof wherein the biologically active fragment is selected from thegroup of consisting of acetyl-transferase (AT) activity; enoyl ACPreductase (ER) activity; dehydrase (DH) activity; malonyl/palmitoylacyltransferase (M/PAT) activity; a first acyl carrier protein (ACP)activity; a second acyl carrier protein (ACP) activity; keto-acyl ACPreductase (KR) activity; keto-acyl ACP synthase (KS) activity; andphosphopantetheinyl transferase (PPT) activity; and b) an amino acidsequence that is at least 85% identical to any of the amino acidsequences of (a) and having the biological activity of the amino acidsequence of (a).
 2. The genetically modified microorganism of claim 1,wherein the nucleic acid molecule encoding the fatty acid synthase is anendogenous gene in the microorganism.
 3. The genetically modifiedmicroorganism of claim 1, wherein the microorganism has been transformedwith the nucleic acid molecule encoding the fatty acid synthase.
 4. Thegenetically modified microorganism of claim 1, wherein the microorganismcomprises an endogenous gene encoding the fatty acid synthase and hasbeen transformed with a recombinant nucleic acid molecule encoding thefatty acid synthase, wherein the gene, and the recombinant nucleic acidmolecule, or both have been modified to increase the expression of thefatty acid synthase.
 5. A biomass comprising the genetically modifiedmicroorganism of claim
 1. 6. A food product or a pharmaceutical productcomprising the biomass of claim
 5. 7. A method to produce short chainfatty acids by a biosynthetic process, comprising culturing in afermentation medium the genetically modified microorganism according toclaim
 1. 8. A genetically modified Thraustochytriales microorganism forproducing short chain fatty acids by a biosynthetic process, wherein themicroorganism comprises multiple copies of a nucleic acid moleculeencoding a fatty acid synthase, wherein the nucleic acid moleculeencoding the fatty acid synthase has been modified to increase theexpression of the fatty acid synthase, and wherein the fatty acidsynthase comprises an amino acid sequence selected from the groupconsisting of: a) an amino acid sequence selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, an amino acid sequence consisting of positions 1-500 of SEQ IDNO:2, an amino acid sequence consisting of positions 450-1300 of SEQ IDNO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ IDNO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ IDNO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ IDNO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ IDNO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ IDNO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ IDNO:2, and a biologically active fragment thereof wherein thebiologically active fragment is selected from the group of consisting ofacetyl-transferase (AT) activity; enoyl ACP reductase (ER) activity;dehydrase (DH) activity; malonyl/palmitoyl acyltransferase (M/PAT)activity; a first acyl carrier protein (ACP) activity; a second acylcarrier protein (ACP) activity; keto-acyl ACP reductase (KR) activity;keto-acyl ACP synthase (KS) activity; and phosphopantetheinyltransferase (PPT) activity; and b) an amino acid sequence that is atleast 85% identical to any of the amino acid sequences of (a) and havingthe biological activity of the amino acid sequence of (a).
 9. Thegenetically modified Thraustochytriales microorganism of claim 1,wherein the amino acid sequence is at least 90% identical to any of theamino acid sequences of (a) and having the biological activity of theamino acid sequence of (a).
 10. The genetically modifiedThraustochytriales microorganism of claim 1, wherein the amino acidsequence is at least 95% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).
 11. The genetically modified Thraustochytriales microorganism ofclaim 1, wherein the amino acid sequence is at least 96% identical toany of the amino acid sequences of (a) and having the biologicalactivity of the amino acid sequence of (a).
 12. The genetically modifiedThraustochytriales microorganism of claim 1, wherein the amino acidsequence is at least 97% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).
 13. The genetically modified Thraustochytriales microorganism ofclaim 1, wherein the amino acid sequence is at least 98% identical toany of the amino acid sequences of (a) and having the biologicalactivity of the amino acid sequence of (a).
 14. The genetically modifiedThraustochytriales microorganism of claim 1, wherein the amino acidsequence is at least 99% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).
 15. The genetically modified Thraustochytriales microorganism ofclaim 8, wherein the amino acid sequence is at least 90% identical toany of the amino acid sequences of (a) and having the biologicalactivity of the amino acid sequence of (a).
 16. The genetically modifiedThraustochytriales microorganism of claim 8, wherein the amino acidsequence is at least 95% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).
 17. The genetically modified Thraustochytriales microorganism ofclaim 8, wherein the amino acid sequence is at least 96% identical toany of the amino acid sequences of (a) and having the biologicalactivity of the amino acid sequence of (a).
 18. The genetically modifiedThraustochytriales microorganism of claim 8, wherein the amino acidsequence is at least 97% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).
 19. The genetically modified Thraustochytriales microorganism ofclaim 8, wherein the amino acid sequence is at least 98% identical toany of the amino acid sequences of (a) and having the biologicalactivity of the amino acid sequence of (a).
 20. The genetically modifiedThraustochytriales microorganism of claim 8, wherein the amino acidsequence is at least 99% identical to any of the amino acid sequences of(a) and having the biological activity of the amino acid sequence of(a).